Columbia  ®nton*ftp 

intljeCttpofltogark 

College  of  3$i}y&itim&  ants  gmrgeong 
lUbrarp 


CLINICAL   PATHOLOGY 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Open  Knowledge  Commons 


http://www.archive.org/details/clinicalpathologOOpant 


CLINICAL  PATHOLOGY 


BY 


P.  N.  PANTON,  M.A.,  M.B.,  B.C.Cantab. 

Clinical  Pathologist  to  the  London  Hospital ;  formerly  Assistant  Director  of  the 
Louis  Joiner  Clinical  Laboratory.  St.  Thomas's  Hospital. 


With  13  Plates  (11  Coloured)  and  45  Illustrations  in  the  Text. 


PHILADELPHIA 

P.   BLAKISTON'S   SON   &   CO. 

1012,   WALNUT    STREET 

1913 


Printed  in  Great  Britain. 


I 

1  <}  -  I  %  ?  1 


PREFACE. 


This  book  represents  an  attempt  to  describe  in  a  reasonable 
compass  such  laboratory  investigations,  whether  chemical, 
histological  or  bacteriological,  as  have  a  practical  bearing 
upon  the  diagnosis  and  treatment  of  disease  ;  to  give  some 
account  of  their  meaning,  and  to  assess,  so  far  as  possible, 
their  value  in  practice. 

Many  of  the  smaller  text-books  of  "Clinical  Pathology" 
deal  with  the  subject  only  in  part ;  the  complexity  of  methods 
in  the  larger  works  renders  them  more  suitable  to  special 
investigators,  and  it  was  felt  that  a  book  of  intermediate 
size  might  be  of  use  to  the  student  and  practitioner.  The 
reduction  in  size  has  been  mainly  arrived  at  by  avoidance  of 
a  reduplication  of  methods,  in  preference  to  the  omission  of 
any  essential  branch  of  the  subject.  Where  several  methods 
for  the  same  object  are  in  use,  one,  or  at  most  two,  are 
described  in  full :  the  remainder  are  omitted  altogether. 

Much  of  scientific  interest  has  been  necessarily  sacrificed  to 
the  practical  application  of  diagnostic  methods,  and  the  book 
has  consequently  no  pretensions  to  consideration  as  anything 
more  than  an  adjunct  to  clinical  medicine. 

No  list  of  references  is  given,  and  the  names  of  authorities, 
unless  definitely  associated  with  a  particular  reaction,  are  for 
the  most  part  omitted. 

In  describing  some  of  the  more  special  investigations  I  have 
made  use  of,  among  other  works,  Sequeira's  "  Diseases  of  the 
Skin,"  Plimmer's  "Practical  Physiological  Chemistry,"  Von 
Jaksch  and  Garrod's  "  Clinical  Diagnosis,"  Sahli's  "  Diagnostic 
Methods,"  and  Daniels  and  Alcock's  "  Tropical  Medicine  and 


vi  PREFACE. 

Hygiene."  The  last  work  has  also  been  of  assistance  to  the 
artist  in  some  of  his  drawings  of  the  higher  parasites. 

I  am  originally  indebted  to  Mr.  L.  S.  Dudgeon  for  many  of 
the  methods  of  procedure  recorded  here  and  for  numerous 
details  of  technique,  which  have  been  of  the  greatest  value  to 
me  in  practice. 

The  illustrations,  with  very  few  exceptions,  have  been 
specially  drawn  for  this  book  from  actual  preparations.  Mr. 
Shattock  has  kindly  provided  me  with  some  of  the  specimens 
of  intestinal  parasites,  and  Dr.  Turnbull  with  others. 

My  colleague,  Dr.  Tidy,  has  most  kindly  read  and  revised 
the  manuscript  and  has  assisted  me  with  his  advice  upon 
numerous  particulars.  Mr.  A.  C.  Hudson  has  also  helped  me 
freely  with  the  final  revision. 


CONTENTS. 


CHAPTER 
I. 
II. 


SECTION   I.— THE    BLOOD 


PAGE 

1—99 


The  Normal  Blood — The  Primary  Blood  Diseases        3 

The    Secondary    Blood    Changes  —  The    Blood 

Changes  occurring  in  Children    .        .        .        .17 

III.    The  Methods  oe  Examining  the  Blood  ...      32 

IY.     The  Blood  Serum — Agglutinins  and  Opsonins      .      48 

V.     The  Blood  Serum  {continued) — Complement  Fixation 

Tests— The  "Wassermann  Beaction        ...       65 
VI.    The  Parasitology  of  the  Blood      .        .        .        .81 
VII.     The  Chemical  and  Physical  Examination  of  the 

Blood 92 


XI 

XII 

XIII 


SECTION   II.— BACTERIOLOGY 


100—188 


VIII.     Introductory — Table  of  Classification  .        .         .101 
IX.     The  Cocci — The  Gram-positive  Bacilli  .        .        .115 
X.     The    Gram-negative    Bacilli — Spirilla — Strepto- 

triche^e — hyphomycetes  .  .  .  .'  .132 
Bacteriological  Methods — General  and  Special.  147 
Vaccines — Anti-sera 164 


Preparation  of  Culture  Media — Staining  Reagents    177 


SECTION   III.— PUNCTURE   FLUIDS  .   189-211 

XIV.    General  Procedure  —  Pleural  Fluids  —  Peri- 
cardial Fluids 190 

XV.    Peritoneal    Fluids  —  Cerebro-spinal    Fluids  - 

Synovial  Fluids — Cysts,  etc 202 


SECTION   IV.— THE   UBINE 


212—284 


XVI.    Routine  Examination  —  Variations  in  Amount  — 

Variations  in  Appearance 213 

XVII.  Variations  in  Acidity,  and  Acidosis — Variations 
in  Specific  Gravity — Urea — Proteids—  Carbo- 
hydrates   228 


viii  CONTENTS. 

CHAPTER  PAGE 

XVIII.     Urinary  Deposits — Urinary  Calculi       .        .        .     248 
XIX.     Special  Investigations  oe  the  Urine—Bacterio- 
logy of  the  Urino-genital  Tract        .        .        .    266 

SECTION   Y.— THE   ALIMENTAEY 

SYSTEM        ....    285-345 

XX.    The  Mouth— The  Stomach 286 

XXI.     The   Pancreas  —  The  Liver  —  The   Spleen  —  The 

Peritoneum 303 

XXII.     The  FiECES 316 

XXIII.  The  Parasitology  of  the  F.eces      ....  329 

SECTION   VI.— THE   EYE   AND    SKIN   346-360 

XXIV.  The  Eye  and  Conjunctival  Sac — The  Skin    .        .    347 

SECTION   VII.— THE.  RESPIRATORY 

TRACT         ....    361-372 

XXV.     The  Nose-  The  Sputum 362 

SECTION  VIII.— HISTOLOGY  .       .   373-433 

XXVI.    The  Examination  of  Sections — The  Inflammations 

— The  Degenerations 374 

XXVII.    Neoplasms — Simple  Tumours 391 

XX  yi  II.     Carcinomata — Sarcomata — Other  Tumours — Cysts  403 

XXIX.    Histological  Methods 421 

INDEX 435 


LIST   OF   PLATES. 


PLATE 

—      ^ 

PAGE 

I. 

Normal  and  Abnormal  Blood  Cells 

To  face         5 

II. 

Pernicious  Anaemia,  Acute  Inflammation 

9 

III. 

11 

IT. 

Lymphoid  Leukaemia         .... 

14 

V. 

Malarial  Parasites 

87 

VI. 

90 

VII. 

Absorption  Spectra 

92 

VIII. 

115 

IX. 

132 

X. 

The  Cells  of  Puncture  Fluids 

192 

XI. 

G-lucosazone  Crystals,  etc.    . 

246 

xn. 

250 

XIII. 

258 

SECTION   I. 

THE    BLOOD. 

CHAPTEE  I. 

The  Normal  Blood — The  Primary  Blood  Diseases. 

CHAPTEE  II. 
The  Secondary  Blood  Changes— The  Blood  Changes  occurring  in  Children. 

CHAPTEE   III. 
The  Methods  of  Examining  the  Blood. 

CHAPTEE   IV. 
The  Blood  Serum — Agglutinins  and  Opsonins. 

CHAPTEE  V. 

The  Blood  Serum  (c.ontin  ued)— Complement  Fixation  Tests— The  "Wasser- 

mann  Eeaction. 

CHAPTEE  VI. 
The  Parasitology  of  the  Blood. 

CHAPTEE  VII. 
The  Chemical  and  Physical  Examination  of  the  Blood. 


CLINICAL    PATHOLOGY. 


CHAPTER  I. 

the  normal  blood — the  primary  blood  diseases. 
The  Normal  Blood. 

The  study  of  the  histology  of  the  blood  in  health  is  properly 
a  part  of  physiology  and  is  essential  to  the  appreciation  of  the 
changes  which  take  place  in  disease.  The  student  is  advised 
to  refresh  his  reading  of  the  normal  blood  before  attempting 
the  study  of  the  blood  in  disease.  The  following  is  a  brief 
account  of  the  normal  blood. 

The  fresh  blood  (page  34) . — If  a  drop  of  blood,  immedi- 
ately after  being  shed,  be  examined  under  the  microscope 
the  red  cells  will  be  found  to  have  arranged  themselves  in  the 
form  of  long  curved  rouleaux,  few  if  any  red  corpuscles  remain- 
ing isolated.  Occasional  leucocytes  will  be  easily  recognised 
lying  between  the  rouleaux,  often  in  groups  of  2  or  3.  Small 
granular-looking  bundles  of  blood  platelets  will  be  made  out, 
usually  in  the  near  neighbourhood  of  a  collection  of  leucocytes. 
After  a  few  minutes  a  delicate  network  of  fibrin,  appearing  like 
a  fine  cobweb,  will  spread  through  the  film,  being  densest  in 
the  vicinity  of  the  blood  platelets.  Later  the  rouleaux  will 
break  up  and  the  individual  red  cells  will  lose  their  shape  and 
become  crenated. 

The  red  corpuscles  are  round,  or  almost  round,  biconcave 
discs  having  an  average  diameter  of  7'5  /x.  They  are  oxyphilic, 
and  in  stained  preparations  the  centre  of  the  disc  is,  owing 
to  the  shape  of  the  cell,  frequently  paler  than  the  periphery. 
The  almost  white  central  area  seen  in  some  preparations  is 
no  evidence  of  disease.  The  number  of  red  cells  is  subject 
to  considerable  individual  variation,  and  is  usually  given  as 
5  million  per  c.mm.  This  number  is  much  below  the  average 
found  in  the  normal  adult  Englishman,  whose  red  cells  vary 


4  CLINICAL   PATHOLOGY. 

between  6  and  7  million  per  c.nini.,  the  number  being  some- 
what less  in  the  case  of  a  woman.  The  number  5  million  is, 
however,  still  commonly  held  to  represent  100  per  cent,  of  the 
normal  for  both  sexes,  mainly  for  the  sake  of  simplicity  in 
estimating  the  colour  index. 

The  non-nucleated  red  cells  of  the  circulating  blood  arise 
from  the  nucleated  cells  of  the  red  marrow.  These  nucleated 
cells  are  of  two  main  varieties,  normoblasts  and  megaloblasts. 
The  former  differ  from  the  circulating  red  cells  in  possessing 
a  single  deeply  and  evenly  staining  nucleus,  which  fills  the 
greater  part  of  the  cell.  The  megaloblast  is  usually  a  larger 
cell,  the  cytoplasm  of  which  always  shows,  though  in  varying 
degree,  an  affinity  for  the  basic  dyes,  the  so-called  basic  or 
polychromatophilic  degeneration.  The  colour  of  the  cytoplasm, 
as  stained  by  Leishman's  stain,  varies  from  a  bronze  to  a 
French  grey  or  even  a  deep  blue,  and  is  probably  evidence,  not 
of  degeneration,  but  of  immaturity.  The  nucleus  is  charac- 
teristically stippled  with  alternating  light  and  dark  areas, 
and  is,  in  a  good  preparation,  readily  distinguished  from  the 
normoblast  nucleus.  The  area  of  cytoplasm  unoccupied  by 
nucleus  is  in  a  megaloblast  relatively  large  (see  Plate  I.). 

The  haemoglobin  is  reckoned  in  percentages,  and  the 
standard  of  the  amount  of  haemoglobin  present  in  a  normal 
person  is  taken  as  100  per  cent.  The  percentage  of  haemo- 
globin varies  very  little  in  health ;  a  fall  below  90  per  cent,  or 
a  rise  above  105  per  cent,  should  be  regarded  as  pathological. 

The  colour  index. — By  this  is  meant  the  index  of  the 
haemoglobin-carrying  properties  of  the  red  cells.  The  index 
is  obtained  by  dividing  the  percentage  of  haemoglobin  by  the 
percentage  of  red  cells  per  c.mm.  For  example,  a  specimen  of 
blood  containing  4  million  red  cells  per  c.mm.,  or  80  per  cent, 
of  the  conventional  normal  number  and  40  per  cent,  of 
haemoglobin,  will  have  a  colour  index  of  f§  or  0-5,  indicating 
that  each  red  cell  contains  half  the  normal  percentage  of 
haemoglobin. 

The  white  corpuscles. — The  normal  number  of  leucocytes 
per  c.mm.  varies  between  5  and  7  thousand,  the  average  number 
being  about  6  thousand.  The  number  of  leucocytes  is  sub- 
ject to  periodic  fluctuations.  The  leucocy  tosis  (i.e.,  increase  in 
the  number  of  leucocytes)  which  occurs  after  meals  is  due  to 
an  increase  in  the  lymphocytes,  and  is  sufficient  to  make  it 


PLATE   I. 


:Cv::v, 


%     #    * 


m 

>tt.'r°i 


tt 


■wr..* 


♦ 


Normal  and  Abnormal  Blood  Cells. 
(Leisb man's  Stain.) 


t 
* 


KEY   TO   PLATE   I. 


MEGALOBL/i57 


OOP 


NORMAL  °     cQ 

<?ED  CELL   BLOOD 

PLATELETS 


The  cells  on  the  right  of  the  vertical  line  are  those 
found  in  normal  blood. 

The  cells  on  the  left  of  the  vertical  line  include  the 
marrow  prototypes  of  the  normal  cells,  and  may  appear 
in  the  blood  in  disease. 

Above  the  horizontal  line  are  the  myeloid  cells,  and 
below,  the  lymphoid  cells. 

The  arrows  indicate  the  probable  cycle  of  cell 
development. 


NORMAL   BLOOD— PRIMARY    BLOOD   DISEASES.    5 

advisable  that  all  blood  examinations  should  be  made  in  the 
intervals  between  meals. 

It  is  essential  to  recognise  that  the  number  of  any  variety 
of  leucocyte  may  alter  both  relatively  to  the  other  varieties  and 
absolutely  in  relation  to  the  total  number  of  leucocytes.  In 
order  to  estimate  the  fluctuations  of  one  variety  its  relative 
number  must  be  considered  in  association  with  the  absolute 
number  of  the  total  leucocytes  and  the  volume  of  the  plasma. 

The  different  varieties  of  leucocytes  are  recognised  by  the 
shape  of  the  nucleus,  the  presence  or  absence  of  granules,  the 
character  of  the  granules  when  present,  and  the  staining 
reaction  of  the  cytoplasm. 

The  following  varieties  are  met  with  (Plate  I.)  : — 

(1)  The  finely  granular  oxyphil,  or  polymorphonuclear 
neutrophil — a  cell  with  a  nucleus  usually  trilobed  and  a 
faintly  basophilic  cytoplasm  thickly  dusted  with  fine,  faintly 
oxyphilic  granules.  The  relative  normal  percentage  of  this 
cell  is  from  60  to  70  of  the  total  leucocytes. 

(2)  The  coarsely  granular  oxyphil,  or  eosinophil— a  cell 
with  a  nucleus  similar  to  the  above  and  a  definitely  basophilic 
cytoplasm  filled  with  large,  strongly  oxyphil  granules.  The 
relative  number  is  from  1  to  3  per  cent. 

(3)  The  coarsely  granular  basophil,  or  mast  cell— a 
cell  with  a  nucleus  usually  bilobed,  a  cytoplasm  which  does 
not  stain  with  the  ordinary  dyes,  and  coarse,  scattered,  strongly 
basophil  granules.     Relative  number,  0  to  1  per  cent. 

(4)  The  finely  granular  basophil— a  cell  similar  to  the 
above,  but  with  fine  basophil  granules  and  with,  as  a  rule,  a 
frayed,  degenerated  nucleus.     Rarely  seen  in  normal  blood. 

(5)  The  large  hyaline  cell,  or  large  mononuclear,  or 
transitional  leucocyte — a  cell  of  the  size  of  a  large  lymphocyte, 
but  with  a  characteristic  notched  or  convoluted  nucleus,  and 
a  reticulated  cytoplasm  which  stains  a  greyish  blue  with 
Leishman's  dye.  This  cell  is  an  important  phagocyte,  is 
amoeboid,  and  is  normally  non-granular.  Relative  number, 
about  5  per  cent. 

(6)  The  large  lymphocyte — a  cell  with  a  round  nucleus 
and  a  clear,  pale  blue  cytoplasm,  which  frequently  contains  a 
few  purple  granules.     Relative  numbers,  5  to  10  per  cent. 

(7)  The  small  lymphocyte— a  cell  with  a  round,  deeply- 
staining  nucleus  which  occupies  almost  the  entire  cell,  leaving 


6  CLINICAL   PATHOLOGY. 

a  narrow  rim  of  deeply  basic  cytoplasm.  Intermediate  forms 
between  the  small  and  the  large  lymphocyte  occur  in  normal 
blood.     Relative  numbers,  20  to  25  per  cent. 

The  origin  of  the  leucocytes  (see  Plate  I.). — It  is 
probable  that  the  first  five  varieties  of  leucocytes  are 
produced  in  the  red  marrow  and  the  lymphocytes  in  the 
lymphoid  tissues,  including  the  small  areas  of  such  tissue 
normally  present  in  the  marrow.  The  lymphocytes  are  pro- 
bably produced  as  such,  the  other  varieties  arising  each  from 
its  special  prototype  in  the  marrow.  The  polymorpho- 
nuclear or,  as  they  are  conveniently  if  not  very  accurately 
called,  polynuclear  neutrophils  arise  from  cells  called  myelo- 
blasts ;  these  are  large  cells  with  round  nuclei  filling  the 
greater  part  of  the  cell  and  a  non-granular,  intensely  baso- 
philic cytoplasm.  The  myeloblasts  give  rise  to  cells  with  a 
round  eccentric  nucleus  and  a  cytoplasm  filled  with  fine,  faintly 
oxyphilic  granules.  They  are  known  as  the  large  type  of 
neutrophilic  myelocyte  (Cornil's  myelocyte).  These  cells 
produce  a  similar  but  smaller  cell  with  a  central  nucleus — the 
small  type  of  neutrophilic  myelocyte  (Ehrlich's  myelocyte). 
The  nucleus  of  this  cell  becomes  notched  and  incompletely 
divided  to  form  the  transitional  polynuclear  neutrophil,  and 
finally  by  further  division  the  polymorphonuclear  neutrophil 
proper. 

All  gradations  of  cells  intermediate  between  the  non-granular 
mononuclear  myeloblast  of  the  marrow  and  the  typical  poly- 
morphonuclear neutrophil  of  the  circulating  blood  can  be  found 
in  the  normal  red  marrow.  The  eosinophil  and  basophil  cells 
are  similarly  produced  from  mononuclear  non -granular  cells, 
which,  developing  the  appropriate  granules,  become  eosinophilic 
or  basophilic  myelocytes.  Some  cells  with  mixed  eosinophil 
and  basophil  granules,  which  ma}"  be  called  amphophilic 
myelocytes,  are  also  occasionally  found. 

The  large  hyaline  appears  to  arise  from  a  marrow  prototype, 
which  has  a  similar  notched  nucleus  and  fine  neutrophilic 
granules  in  its  cytoplasm  ;  such  granules  may  occasionally  be 
detected  in  the  hyalines  of  the  normal  blood. 

An  acquaintance  with  the  probable  mode  of  formation  of  the 
normal  leucocytes  and  red  cells  is  necessary  for  an  appreciation 
of  the  changes  which  may  take  place  in  the  circulating  blood 
in  disease,  since  under  the  appropriate  stimulus  the  cells  of 


NORMAL   BLOOD— PRIMARY   BLOOD   DISEASES.     7 

the  marrow  may  overflow  into  the  peripheral  blood-stream.  A 
moderate  stimulus  to  the  red  cell  formation,  as  after  haemor- 
rhage, may  lead  to  the  appearance  of  occasional  normoblasts  in 
the  blood.  An  intense  stimulus,  as  in  pernicious  anaemia,  may 
bring  megaloblasts  into  the  blood  in  considerable  numbers.  A 
moderate  over-production  of  leucocytes,  as  in  acute  inflamma- 
tion, may  be  accompanied  by  the  appearance  of  transitional 
neutrophils  and  the  small  type  of  myelocytes.  An  extreme 
over-production,  as  in  myeloid  leukaemia,  throws  into  the  cir- 
culating blood  every  variety  of  marrow  leucocyte,  including 
the  mononuclear  non-granular  myeloblasts. 

In  addition  to  the  various  cells  in  a  stained  preparation 
of  normal  blood,  the  blood  platelets  can  be  recognised  as  tiny 
basophilic  rounded  bodies  of  somewhat  irregular  shape, 
occurring  either  singly  or  in  small  clumps  throughout  the  film. 
The  platelets  are  possibly  derived  from  the  leucocytes,  and  are 
possibly  nothing  more  than  a  granular  deposit  taking  place  in 
the  shed  blood.  Their  significance  and  mode  of  formation  are 
obscure.  A  single  platelet  lying  on  a  red  corpuscle  is 
frequently  mistaken  for  a  malarial  parasite,  which  it  does  not 
particularly  resemble. 

Pkimaey  Blood  Diseases. 

By  primary  blood  affections  are  meant  diseases  accompanied 
by  characteristic  changes  in  the  blood  and  with  no  recognised 
primary  lesion  elsewhere  in  the  body.  It  does  not  follow,  and 
is  indeed  improbable,  that  the  so-called  primary  blood  diseases 
are  essentially  diseases  of  the  blood  or  blood-forming  organs. 

These  primary  blood  diseases  are  four  in  number,  of  which 
two  affect  mainly  the  erythroblastic  and  two  the  leucoblastic 
mechanism.  The  red  cell  elements  may  be  affected  in  one 
of  two  ways,  either  in  their  morphology  or  in  their  haemo- 
globin -  carrying  capacity.  The  white  cell  elements  may 
likewise  be  affected  either  in  their  myelogenic  or  in  their 
lymphocytic  modes  of  production.  These  four  varieties  of 
affection  constitute  the  four  primary  blood  diseases — Pernicious 
Anaemia,  Chlorosis,  Myeloid  Leukaemia,  Lymphoid  Leukaemia. 

If  it  be  remembered  that  a  disease  which  primarily  affects 
the  erythroblastic  function  of  the  marrow  affects  also  to  a  less 
degree  the  leucoblastic  element  and  vice  versa,  the  main  blood 


8  CLINICAL   PATHOLOGY. 

changes  in  any  of  the  primary  diseases  are  with  the  assistance 
of  the  following  simple  scheme  easily  remembered. 

The  Blood. 


The  red  cells.                                            The  white  cells. 
I I 


Hemoglobin  Morphology.  Granular  Xon-granular 

content.  (myelogenous).         (lymphogenous). 

Chlorosis.  Pernicious         Myeloid  leukaemia.  Lymphoid 

anaemia,  leukaemia. 

Chlorosis  is  a  common  affection  almost  exclusively  corn- 
fined  to  young  unmarried  girls.  The  cause  of  the  disease  is 
unknown,  but  is  probably  dependent  upon  some  abnormal 
state  of  the  female  reproductive  organs.  The  condition 
readily  improves  with  treatment,  but  a  return  of  the  blood  to 
normal  is  always  slow  and  rarely  complete  unless  a  radical 
alteration  is  made  in  the  surroundings  or  mode  of  life  of  the 
patient. 

The  main  alteration  detected  by  the  routine  examination  of 
the  blood  is  a  loss  in  haemoglobin.  In  a  case  of  moderate 
severity  there  is  little  diminution  in  the  number  of  red  cells, 
consequently  the  colour  index  is  extremely  low.  In  such 
a  case  the  haemoglobin  would  be  about  40  per  cent.,  the  red 
cells  about  4  million  per  c.mm.,  and  the  colour  index  0'5.  In 
a  severe  case  the  haemoglobin  percentage  falls  very  low  indeed, 
and  the  red  cells  may  be  considerably  reduced,  though  rarely 
to  a  number  less  than  3  million  per  c.mm.  In  such  a  case 
occasional  normoblasts  and  misshapen  red  cells  or  poikilocytes 
may  be  found  in  addition.  The  leucocytes  are  not  altered  in 
any  characteristic  manner,  but  they  may  be  slightly  diminished, 
and  not  infrequently  the  lymphocytes  are  relatively  increased. 
In  all  cases  the  total  blood  volume  is  considerably  increased, 
and  the  oxygen  capacity  remains  about  normal,  the  increase 
in  volume  being  due  to  an  increase  in  the  amount  of  plasma. 

Pernicious  anaemia  (Idiopathic,  Progressive,  Addisonian 
anaemia)  is  a  rare  and  fatal  disease  affecting  both  sexes. 
It  is  rather  more  common  in  males  than  in  females,  and 
may  occur  at  any  age,  being  most  frequent  in  patients 
aged  between  35  and  45  years.  The  average  course  of 
pernicious  anaemia  is  two  years,  and  remarkable  remissions 


PLATE  II. 


PLATE   II. 


Pernicious  ArtEemia. 
(Irishman's  Stain.) 


Acute  Inflammation. 
(Irishman's  Stain.) 


NORMAL  BLOOD— PRIMARY  BLOOD  DISEASES.  9 

in  the  severity  of  the  disease  are  the  rule.  During  these 
remissions  the  blood  may  return  to  normal.  No  constant 
underlying  factor  has  yet  been  discovered  in  pernicious 
anaemia,  and  all  that  is  known  is  that  the  erythroblastic 
marrow  is  abnormally  active,  that  abnormal  red  cells  are 
present  in  the  circulating  blood,  and  that  the  destruction 
of  red  cells  in  the  tissues  is  excessive.  On  clinical  grounds 
the  disease  may  be  strongly  suspected  in  a  markedly  anaemic 
patient  complaining  of  extreme  weakness  without  other 
symptoms  or  physical  signs  to  account  for  his  prostration. 
The  diagnosis  can  be  confirmed  only  by  careful  and,  if 
necessary,  repeated  examination  of  the  blood.  The  examina- 
tions, to  be  of  any  value,  must  be  complete  and  accurate, 
every  abnormal  change  being  judiciously  estimated  before  an 
accurate  diagnosis  can  be  made.  One  has  been  asked  by  able 
practitioners  to  make  a  diagnosis  from  blood  films  alone,  which 
is  rarely  advisable,  and  even  from  blood  daubed  on  pieces  of 
window  glass  or  other  amateur  contrivances,  which  is  impos- 
sible. 

The  blood  changes  are  as  follow  : — 

The  drop  of  blood  as  it  exudes  from  the  puncture  is  exces- 
sively liquid  and  has  a  peculiar  streaked  appearance.  The 
colour  is  noticeably  brighter  than  would  be  expected  from  the 
pallor  of  the  patient.  If  some  blood  is  collected  in  a  capsule 
and  allowed  to  stand,  the  serum  separates  with  marked 
rapidity,  leaving  a  disproportionately  small  column  of  red  cells. 
The  colour  of  the  supernatant  serum  is  a  curious  greenish 
yellow,  a  colour  scarcely  seen  in  any  other  disease.  The 
fresh  blood  under  the  microscope  shows  absence  of  rouleaux 
formation,  with  a  marked  tendency  of  the  red  cells  to  come 
together  into  clumps  of  considerable  size.  It  is  this  clumping 
of  the  red  cells  which  gives  the  streaked  appearance  to 
tbe  fresh  drop.  Many  of  the  red  cells  are  altered  in 
shape  (poikilocytosis),  others  in  size,  the  large  cells  or 
macrocytes  being,  as  a  rule,  more  numerous  than  the  small 
cells  or  microcytes.  Some  of  the  cells  may  be  seen  to  put  out 
processes  and  to  undergo  apparent  amoeboid  movements. 
Fibrin  formation  is  slight  or  absent  and  blood  platelets  are 
few  in  number. 

The  red  cells  are  greatly  diminished,  usually  to  a  number  in 
the  near  neighbourhood  of  1  million  per  c.mm. 


10  CLINICAL   PATHOLOGY. 

The  haemoglobin  is  much  diminished,  but  not  in  proportion 
to  the  decrease  in  the  erythrocytes,  so  that  the  colour  index  is 
high  and  usually  varies  between  0-8  and  1*4.  In  a  typical 
case  the  red  cells  would  be  1  million  per  c.mm.,  the 
haemoglobin  25  per  cent.,  and  the  colour  index  1'25. 

The  leucocytes  are  almost  invariably  decreased  to  between 
3  and  4  thousand  per  c.mm. 

In  the  stained  blood  the  more  important  changes  are  found 
in  the  red  cells,  many  of  which  are  found  to  be  abnormal  in 
shape  and  size.  These  deformities,  however,  may  be  artificially 
produced  in  the  preparation  of  stained  sjDecirnens,  and  should 
always  be  confirmed  by  an  examination  of  the  fresh  blood. 
Nucleated  red  cells,  both  normoblasts  and  megaloblasts,  are 
usually  present  and  may  be  very  numerous :  of  these  the 
former  are  found  in  any  variety  of  severe  anaemia ;  the 
megaloblasts  are  practically  never  seen  in  any  other  condition, 
with  the  exception  of  the  leukaemias.  The  recognition  of  the 
megaloblast  is  therefore  of  importance,  and  depends  less  upon 
the  size  of  the  cell  than  on  the  character  of  the  cytoplasm, 
and  particularly  upon  the  mottled  stippling  of  the  nucleus.  In 
some  cases  before  treatment  nucleated  red  cells  may  be  absent, 
and  may  appear  in  considerable  numbers  after  the  patient  has 
been  given  arsenic.  Polychroinatophilic  cells  are  usually 
numerous,  and  other  red  cells  are  present  which  show  basic 
granules  in  their  cytoplasm — the  so-called  granular  degenera- 
tion. Certain  other  but  less  noticeable  changes  are  present 
in  the  leucocytes,  namely,  a  relative  increase  in  the  small 
lymphocytes  and  to  a  less  extent  in  the  eosinophil  cells. 
Occasional  mast  cells  may  be  present  together  with  2  or  3  per 
cent,  of  neutrophilic  myelocytes,  usually  of  the  small  type.. 

The  majority  of  the  changes  described  above  may  occur  in 
any  of  the  secondary  anaemias  if  sufficiently  intense,  the  two 
changes  most  characteristic  of  pernicious  anaemia  being  the 
high  colour  index  with  a  low  red  cell  count  and  the  presence 
of  megaloblasts. 

The  conditions  most  liable  to  be  mistaken  for  pernicious 
anaemia  are  those  due  to  certain  known  organic  causes  which 
are  capable  of  producing  a  severe  anaemia,  and  which  may  on 
occasion  fail  to  give  their  proper  physical  signs  and  symptoms. 
Such  are  latent  carcinoma,  and  in  particular  carcinoma  of 
the  stomach,  Addison's  disease,  intestinal  parasites,  and  the 


PLATE    I  [I. 


PLATE   III. 


Myeloid  Leukaemia. 
(Typical  Film  from  a  case  of  2  years'  duration.)     (Irishman's  Stain.) 


Myeloid  Leukeemia. 
(Myeloblasts  Transformation  ;  from  the  same  case  as  the  above,  1  year  later 
and  1  week  before  death.) 
(Leishman's  Stain.) 


NORMAL   BLOOD— PRIMARY   BLOOD   DISEASES.    11 

anaemias  following  severe  or  small  and  repeated  haemorrhages, 
particularly  when  occurring  in  puerperal  women.  Pernicious 
anaemia  is  sometimes  associated  with  definite  lesions  in  the 
spinal  cord,  and  these  may  be  of  the  type  and  distribution 
known  as  posterior  lateral  sclerosis.  The  same  nerve  lesions 
may  in  other  patients  be  associated  with  an  anaemia  of  the 
secondary  type. 

The  diagnosis  of  pernicious  anaemia  may  be  difficult.  After 
a  careful  examination  of  the  patient  and  of  his  blood  mistaken 
diagnoses  should  be  extremely  rare. 

Aplastic  anaemia  may  be  regarded  as  a  form  of  pernicious 
anaemia  in  which  the  marrow  has  failed  to  react.  Haemoglobin, 
red  cells  and  white  cells  are  all  greatly  reduced,  the  marrow  is 
atrophied,  and  nucleated  red  cells  are  absent.  The  disease 
is  of  great  rarity,  usually  affects  young  individuals,  and  is 
rapidly  fatal. 

Myeloid  leukaemia  (Myelogenous  leukaemia,  Myelaemia, 
Spleno-medullary  leukaemia). — This  disease,  somewhat  less 
rare  than  pernicious  anaemia,  affects  males  more  frequently 
than  females,  the  average  age  of  incidence  being  from  15  to  30 
years.  Myeloid  leukaemia  may  run  an  acute  course  of  a  few 
weeks  or  months ;  it  is,  however,  very  much  more  frequently 
a  comparatively  chronic  disease,  which  terminates  fatally  in 
about  2  years.  The  early  symptoms  are  trivial  and  usually 
due  to  the  great  size  of  the  spleen,  a  size  only  met  with  in  this 
country  in  splenic  polycythaemia  and  in  the  varieties  of  splenic 
anaemia.  Myeloid  leukaemia  can  very  readily  be  diagnosed  by 
an  examination  of  the  blood.  The  blood  changes  are  as  follow 
(Plate  III.):  — 

The  blood  flows  readily  on  puncture,  and  may  continue  to 
ooze  for  some  hours.  The  fresh  blood  under  the  microscope 
is  quite  characteristic  ;  the  leucocytes  are  so  numerous  that 
they  appear  to,  but  practically  never  do,  outnumber  the  red 
cells.  The  majority  of  the  cells  are  seen  to  be  granular,  the 
retractile  granules  showing  quite  well  in  the  unstained  blood. 
The  red  cells  as  a  rule  show  little  change,  fibrin  formation  is 
present,  and  the  blood  platelets  are  extremely  numerous  and 
massed  into  large  clumps. 

The  leucocytes  are  enormously  increased,  the  usual  number 
found  in  an  untreated  case  being  about  200,000  per  c.mm. 
Such    extreme   leucocytosis    is    hardly    seen    in    any    other 


12  CLINICAL   PATHOLOGY. 

condition.  The  red  cells  and  haemoglobin  are  both  diminished, 
slightly  in  the  early  stages,  markedly  in  the  later  periods  of 
the  disease.     The  colour  index  is  moderately  low. 

The  stained  blood  film  is  prepared  with  some  little  difficulty 
owing  to  the  large  number  of  leucocytes  which  necessitates 
the  obtaining  of  a  particularly  thin  film  without  pressure. 
As  the  film  dries  it  presents  a  curious  greasy,  semi-opaque 
appearance  due  to  the  leucocytosis.  Over  90  per  cent,  of  the 
leucocytes  are  found  to  be  granular  and  to  consist  of  the 
following  varieties : — 

Polynuclear  neutrophils    .         .     about  40  to  50  per  cent. 

Transitional  neutrophils  . 

Neutrophil  myelocytes  (both  small  and 
large)    ..... 

Eosinophils       .... 

Eosinophil  myelocytes 

Finely  granular  basophil  cells  . 

Mast  cells  .... 

Basophil  myelocytes 

Amphophil  myelocytes 
A  proportion  of  the  non-granular  cells 

but  many  intermediate  varieties  of  cells,  which  may  be  difficult 
to  classify,  are  met  with.  Nucleated  red  cells,  both  normo- 
blasts and  megaloblasts,  are  usually  found,  and  may  be 
particularly  numerous  in  cases  with  severe  anseinia  or  after 
a  hasrnorrhage — a  not  infrequent  occurrence  in  this  disease. 

The  most  important  of  the  blood  changes  are — the  great 
leucocytosis ;  the  enormous  preponderance  of  granular  cells  ; 
the  absolute  increase  of  mast  cells  and  eosinophil  cells ;  the 
appearance  of  marrow  prototypes  in  the  blood  ;  the  presence 
of  nucleated  red  cells. 

The  blood  picture  of  myeloid  leuksemia  may  be  completely 
altered  by  treatment,  particularly  by  the  administration  of 
arsenic  or  the  application  of  X  rays.  The  most  common 
results  of  treatment  consist  in  a  diminution  in  the  size  of 
the  spleen,  accompanied  by  a  decrease  in  the  number  of 
leucocytes.  In  some  cases  the  spleen  disappears  beneath  the 
costal  margin  and  the  leucocytes  fall  to  the  normal  number 
or  even  below  the  normal.  In  such  circumstances  the  stained 
film  may  show  little  evidence  of  disease,  but  as  a  rule  occa- 
sional myelocytes  persist  and  the  mast  cells  remain  relatively 


about  10 

>> 

i>nd 

.      20  to  30 

j) 

5  to  10 

5  J 

2  to    5 

) ; 

"J     5  to  20 

55 

2  to    5 

)j 

1  to    3 

55 

consists  of  n 

a    wVi  i  r».Vi  m  a.\j 

iyeloblasts, 

1->a  rliffip.nlf, 

NORMAL   BLOOD—PRIMARY   BLOOD   DISEASES.    13 

increased.  Commonly  the  fall  in  the  number  of  leucocytes  is 
more  marked  than  the  shrinkage  of  the  spleen,  and  a  patient 
may  have  a  leucopenia  with  a  spleen  reaching  to  the  umbilicus. 
Such  a  condition  might  be  mistaken  for  splenic  anaemia. 
These  changes  are  favourable  and  may  persist  for  some 
months  before  a  relapse  occurs. 

In  the  terminal  stages  of  the  disease  a  diminution  in  the 
number  of  leucocytes  may  be  accompanied  by  the  appearance 
of  large  numbers  of  non-granular  cells  in  the  blood.  These 
non-granular  cells  are  myeloblasts  and  in  their  more  usual 
type  are  of  about  the  size  of  large  lymphocytes,  but  differ 
from  them  in  having  a  relatively  large  nucleus,  containing 
3  or  4  pale  pear-shaped  nucleoli  and  an  intensely  basophilic 
cytoplasm  (Plate  III.).  A  less  common  type  of  myeloblast  is 
little  larger  than  the  small  lymphocyte,  and  in  all  probability 
represents  a  non-granular  stage  of  the  small  type  of  neutro- 
philic myelocyte  ;  it  is  distinguished  from  the  small  lympho- 
cyte by  the  purplish  colour  of  its  cytoplasm.  Both  these 
forms  of  myeloblast  are  to  be  found  in  small  numbers  in  all 
stages  of  the  disease.  A  marked  increase  in  their  relative 
numbers  is  of  the  gravest  significance. 

Acute  myeloid  leukaemia  is  an  extremely  rare  condition, 
which  differs  from  the  ordinary  form  of  the  disease  in  the 
shortness  of  its  course,  in  the  usual  absence  of  marked  splenic 
enlargement,  and  in  the  relatively  low  leucocytosis.  There 
may  be  considerable  involvement  of  the  lymphatic  glands. 
Mast  cells,  eosinophils,  and  myelocytes  are  present,  but  the 
predominant  cell  may  be  either  a  myeloblast  or  a  cell  identical 
with  the  large  hyaline  of  normal  blood  or  a  granular  variety 
of  it. 

Lymphoid  leukaemia  (Lymphatic  leukaemia,  Lymphaemia), 
the  least  common  of  the  primary  anaemias,  may  occur  at  any 
age,  but  usually  attacks  children  between  the  ages  of  8  and  15. 
In  its  typical  form  lymphaemia  is  an  acute  disease,  accompanied 
by  fever  and  severe  constitutional  disturbance,  and  terminating 
fatally  in  a  few  weeks.  There  is  a  moderate  general  enlarge- 
ment of  the  superficial  lymphatic  glands  and  the  spleen  as  a 
rule  is  readily  palpable.     The  blood  changes  are  as  follow : — 

The  blood  flows  readily  on  puncture  and  the  excess  of 
leucocytes  is  very  obvious  in  the  fresh  drop  under  the  micro- 
scope.    The  leucocytes  are  seen  to  be  non-granular.     Fibrin 


14  CLINICAL   PATHOLOGY 

formation  is  slight  and  blood  platelets  are  very  scanty.  The 
leucocytes  are  much  increased,  being  commonly  about  60,000 
per  c.mm.,  a  number  intermediate  between  that  found  in  acute 
inflammation  and  in  myeloid  leukaemia. 

The  red  cells  and  haemoglobin  are  as  a  rule  greatly 
diminished,  and  often  to  the  extent  found  in  pernicious 
anaemia.  The  colour  index  not  infrequently  remains  rela- 
tively high,  and  may  be  above  the  normal. 

The  stained  film  is  very  characteristic,  almost  the  entire 
leucocytes  consisting  of  lymphocytes.  The  lymphocytes  are 
in  the  majority  of  cases  of  the  small  variety,  but  differ  in 
some  respects  from  those  of  normal  blood.  They  are  as  a 
rule  larger  than  the  normal  cell,  but  retain  the  relatively 
small  proportion  of  cytoplasm  to  nucleus.  The  cytoplasm 
has  the  same  staining  reaction  as  that  of  the  normal  cell ;  the 
nucleus  is  as  a  rule  atypical,  taking  the  basic  dye  less  deeply 
than  normal  and  being  frequently  notched  or  indented  after 
the  fashion  of  the  nucleus  of  the  large  hyaline.  Less 
commonly  the  lymphocyte  is  of  the  large  type,  and  both 
varieties,  together  with  intermediate  forms,  may  occur  in  the 
same  film.  Some  authorities  consider  these  atypical  cells  to 
be  non-granular  myelocytes  produced  in  the  bone  marrow  and 
not  of  lymphoid  origin.  The  relative  proportions  of  large  and 
small  lymphocytes  may  fluctuate  greatly  from  day  to  day.  The 
total  lymphocytes  usually  comprise  from  90  to  99  per  cent,  of 
all  the  leucocytes.  The  red  cells  as  a  rule  show  the  changes 
usual  in  any  severe  secondary  anaemia,  but  nucleated  red  cells 
are  frequently  absent.  Both  normoblasts  and  megaloblasts, 
may  however,  be  found,  particularly  after  a  severe  haemorrhage. 

The  most  important  of  the  blood  changes  in  lymphoid 
leukaemia  consist  in  a  great  increase  in  the  total  number  of 
leucocytes  accompanied  by  an  almost  complete  replacement  of 
the  normal  cells  by  atypical  lymphocytes,  usually  of  the  small 
variety.  The  variety  of  lymphocyte  present  is  no  guide  to  the 
acuteness  of  the  disease. 

The  blood  changes  are  practically  diagnostic ;  it  must  be 
remembered,  however,  that  a  very  high  degree  of  lymphocy- 
tosis is  known  to  occur  in  quite  small  children,  apart  from 
this  disease.  The  blood  of  children  below  the  age  of  5  has 
normally  a  relatively  high  number  (40  to  50  per  cent.)  of 
small  lymphocytes,  and  these  cells  are  the  ones  most  liable  to 


PLATE   IV. 


Acute  Lymphoid  Leukasmia. 
(Lymphocytes  of  Small  Type.)     (Leishman's  Stain.) 


Chronic  Lymphoid  Leukasmia. 

(Lymphocytes  of  Large  Type  ;  from  a  case  of  3  years'  duration.) 

(Leishman's  Stain.) 


PLATE   IV. 


NOKMAL   BLOOD— PRIMARY   BLOOD   DISEASES.    15 

increase  in  several  varieties  of  diseased  conditions,  and  parti- 
cularly during  an  attack  of  whooping  cough. 

Chronic  lymphoid  leukaemia  is  an  extremely  rare  con- 
dition, resembling  in  its  clinical  aspects  nryelaeinia  rather 
than  lymphgemia.  The  spleen  may  be  greatly  enlarged  and 
the  glands  little  if  at  all  affected.  The  blood  condition 
resembles  that  of  the  acute  disease,  and  the  lymphocytes  may 
be  of  the  small  or  of  the  large  variety. 

Chloroma  is  in  all  probability  a  variety  of  acute  lymphoid 
leukaemia,  and  the  blood  picture  may  be  identical.  Less  com- 
monly the  blood  changes  are  myeloid  in  character.  In  addition 
to  the  clinical  features  of  lymphgemia  the  patient  presents  a 
peculiar  greenish  coloration  of  the  skin,  together  with 
evidence  of  subperiosteal  swellings,  most  numerous  on  the 
bones  of  the  skull.  Bilateral  proptosis  is  usual.  The  sub- 
periosteal infiltrations  are  of  a  bright  green  colour,  which  fades 
on  exposure  to  the  air.  The  green  pigment  is  not  present  in 
all  cases,  and  is  not  confined  to  this  condition.  It  may  be 
found  in  the  lymph  glands  in  some  cases  of  myelaemia. 
Chloroma  is  regarded  by  some  authorities  as  a  form  of  new 
growth  of  the  nature  of  a  sarcoma,  and  the  blood  condition  is 
considered  to  be  due  to  a  leakage  of  the  malignant  cells  from 
the  tumours  into  the  circulation.  In  the  usual  forms  of 
sarcoma  no  such  leakage  of  tumour  cells  appears  to  occur. 
The  disease  seems  to  affect  especially  children  of  the  Hebrew 
race. 

Summaey. 

An  acquaintance  with  the  normal  blood  is  essential  before 
undertaking  the  diagnosis  of  disease,  and  students  are  advised 
to  examine  the  blood  of  patients  not  suspected  of  blood 
changes  in  order  to  appreciate  the  variations  in  the  normal. 
The  blood  of  normal  individuals  differs  just  as  the  normal 
breath  sounds  differ,  and  an  acquaintance  with  these  variations 
renders  the  recognition  of  diseased  conditions  certain. 

The  derivation  of  all  leucocytes  from  cells  of  one  type  in 
the  adult  is  possible  but  not  proven. 

The  simplest  classification  of  the  normal  leucocytes  is  into 
the  non-granular  mononuclear  cells  of  the  lymphoid  system 
and  the  polynuclear  cells  of  the  myeloid  system. 

The  primary  anaemias  are  diseases  associated  with  striking 


16  CLINICAL   PATHOLOGY. 

changes  in  the  blood  of  which  the  causes  are  unknown.  With 
the  exception  of  chlorosis  these  are  rare  diseases.  The 
diagnosis  in  all  of  them  can  be  made  with  tolerable  certainty 
by  means  of  an  examination  of  the  blood,  provided  that  the 
fluctuating  nature  of  the  blood  changes  are  recognised  and 
that  every  advantage  is  taken  of  the  clinical  information 
derived  from  the  patient. 


CHAPTER    II. 

THE    SECONDARY    BLOOD    CHANGES — THE    BLOOD    CHANGES 
OCCURRING    IN    CHILDREN. 

A  description  of  the  changes  which  may  be  found  by  means 
of  an  ordinary  blood  examination  in  every  known  disease 
would  be  obviously  impossible,  and  indeed  is  unnecessary,  since 
similar  pathological  conditions  underlie  different  diseases  and 
give  rise  to  similar  changes  in  the  blood. 

The  secondary  blood  changes  and  their  diagnostic  signifi- 
cances are  conveniently  described  under  the  following  five 
headings,  which  denote  the  predominant  change  associated 
with  various  conditions : — 

(1)  An  increase  in  the  number  of  white  cells,  or 
leucocytosis. 

(2)  An  increase  in  the  number  of  eosinophil  cells,  or 
eosinophilia. 

(3)  A  decrease  in  the  number  of  white  cells,  or  leucopenia. 

(4)  An  increase  in  the  number  of  red  cells,  or 
polycythemia. 

(5)  A  decrease  in  the  number  of  red  cells,  or  oligocythemia. 

(1)  Conditions  associated  with  a  leucocytosis. 

Acute  inflammation  is  the  most  important  underlying 
cause  of  an  increase  in  the  white  cells  in  the  secondary  blood 
diseases.  A  typical  example  of  the  acute  inflammatory 
process  set  up  by  one  of  the  pyogenic  organisms  is  met  with 
in  lobar  pneumonia.  The  blood  changes  present  in  this  con- 
dition are  as  follow : — 

The  blood  clots  readily,  and  an  increase  in  the  blood  platelets, 
together  with  an  excessive  fibrin  formation,  is  seen  in  the 
fresh  blood.  A  comparatively  mild  secondary  ansemia  is 
usually  present.  The  leucocytes  are  markedly  increased,  often 
up  to  20  or  30,000  per  c.mm.  The  number  of  the  leucocytes 
does  not  fall  with  the  temperature,  but  gradually  diminishes  as 

p.  2 


18  CLINICAL   PATHOLOGY. 

resolution  occurs  in  the  lung.      The  stained  film  (Plate  II., 
yields  the  following  differential  count : — 

Polynuclear  neutrophils      .         .     80  to  95  per  cent. 
Eosinophils         ....     absent. 
Large  hyalines    .         .         .         .     5  to  10  per  cent. 
Small  and  large  lymphocytes       .     2  to  10  per  cent. 

The  leucocytosis  is  seen  to  be  due  mainly  to  an  absolute 
and  relative  increase  in  the  polynuclear  neutrophils  and  to 
a  lesser  degree  to  an  absolute  increase  in  the  phagocytic 
hyaline  cells. 

The  complete,  or  almost  complete,  absence  of  the  eosinophils 
is  a  very  constant  feature,  and  the  reappearance  of  these  cells 
is  an  indication  that  the  inflammatory  process  is  undergoing 
resolution. 

Very  occasionally  in  acute  inflammation  there  is  no  increase 
in  the  leucocytes,  and  they  may  even  be  diminished  ;  the 
relative  proportion  of  the  leucocytes,  however,  is  altered  in  the 
manner  described  above.  In  a  fatal  case  of  extensive  sup- 
puration in  the  bile  passages  the  total  number  of  leucocytes 
was  only  3,000  per  c.mni.,  but  the  polynuclear  neutro- 
phils formed  90  per  cent,  of  the  white  cells.  Such  a  blood 
picture  occurs  in  a  patient  whose  protective  mechanism  is 
failing  to  react  to  the  infection,  and  is  of  the  gravest 
prognosis. 

The  blood  changes  of  lobar  pneumonia  are  similar  to  those 
in  all  acute  inflammatory  processes  set  up  by  any  of  the 
pyogenic  organisms,  such  as  the  streptococci,  staphylococci, 
colon  bacillus  and  the  like,  and  are  of  assistance  in  arriving  at 
a  clinical  diagnosis.  In  a  case  of  doubtful  appendicitis  an 
inflammatory  blood  count  means  that  there  is  acute  inflamma- 
tion in  the  body,  not  necessarily  in  the  appendix.  It  does  not 
mean  that  actual  suppuration  has  taken  place  and  that 
immediate  surgical  interference  is  necessary  on  that  account. 
The  processes  of  acute  inflammation  and  suppuration  differ 
only  in  degree,  and  so  do  the  changes  in  the  blood.  When  pus 
is  pent  up  in  the  body  the  leucocytes  tend  to  increase ;  wrhen 
inflammation  is  resolving  the  white  cells  diminish  and  the 
eosinophils  reappear.  A  rising  leucocyte  count  in  a  case  of 
appendicitis  is  evidence  of  abscess  formation ;  a  falling  count 
with  a  diminution  in  the  relative  number  of  polynuclear 
neutrophils  is  evidence  of  resolution.     The  clinical  value  of 


THE    SECONDARY   BLOOD   CHANGES.  19 

the  blood  examination  therefore  depends  not  only  upon  an 
enumeration  of  the  total  number  of  leucocytes  present,  but 
also  upon  an  estimation  of  the  relative  percentage  of  the  cells 
found  in  the  stained  blood,  and  in  addition  a  series  of  examina- 
tions may  be  necessary. 

Fevers. — Among  the  pathological  processes  giving  similar 
changes  in  the  blood  are  certain  fevers  of  unknown  etiology, 
such  as  scarlet-fever,  small-pox,  chicken-pox  and  rheumatic 
fever. 

In  carcinoma,  particularly  in  carcinoma  of  the  intestine, 
an  inflammatory  blood  count  is  usual,  and  is  probably  due  to 
the  infective  processes  set  up  by  the  growth. 

In  tuberculosis,  in  its  usual  and  comparatively  localised 
forms,  the  blood  changes  are  very  different,  but  in  generalised 
tuberculosis,  especially  with  wide  spread  glandular  involvement, 
a  well-marked  inflammatory  count  is  common.  If  a  patient 
with  general  glandular  enlargement  is  found  to  have  a  marked 
leucocytosis  of  the  polynuclear  variety  the  condition  is 
extremely  likely  to  be  tuberculous. 

The  presence  of  an  inflammatory  leucocytosis  is  always 
strongly  suggestive  of  one  of  the  above  diseases,  and  helps  to 
distinguish  them  as  a  group  from  any  of  the  affections 
commonly  associated  with  a  leucopenia,  such  as  influenza  or 
typhoid  fever.  A  leucocytosis  occurring  in  typhoid  fever 
indicates  that  some  complication  is  present — for  example,  an 
infection  of  the  bile  passages.  A  continuous,  high  and  rising 
leucocytic  count  suggests  that  an  inflammatory  process  has 
gone  on  to  pus  formation.  A  low  total  count  with  a  high 
relative  number  of  polynuclear  neutrophils  is  of  grave 
prognosis. 

While  an  inflammatory  leucocytosis  is  found  with  all  acute 
septic  lesions,  an  apparent  exception  must  be  referred  to  in 
tropical  abscess  of  the  liver.  In  this  condition,  particularly 
when  latent,  leucocytosis  is  frequently  absent ;  but  it  must  be 
remembered  that  the  "  abscess "  is  in  reality  a  chronic 
necrotic  condition  and  does  not  contain  pus.  If  infection  by 
pyogenic  organisms  occurs  in  the  necrotic  liver  a  leucocytosis 
supervenes. 

(2)  Conditions  associated  with  an  eosinophilia. 

Animal  parasites  are  among  the  most  important  agents 

2—2 


20  CLINICAL   PATHOLOGY. 

capable  of  producing  an  increase  in  the  eosinophil  cells.  Any  of 
the  common  intestinal  worms  may  cause  an  eosinophilia,  and 
in  particular  the  ankylostoma,  which  produces  in  addition  a 
severe  anaemia  of  the  secondary  type.  Trichinosis,  hydatid 
disease,  filariasis,  and  bilharzia  disease  also  produce  an 
eosinophilia.  The  increase  in  the  eosinophil  cells  is  usually 
accompanied  by  a  leucocytosis,  and  the  relative  percentage 
of  the  eosinophils  may  vary  from  5  to  60  per  cent.,  or  even 
more,  so  that  the  total  increase  in  these  cells  may  be  very 
considerable.  In  the  case  of  hydatid  disease  the  eosinophilia 
may  persist  even  when  the  cyst  has  become  fibrous  and 
partly  calcined.  With  cysts  which  have  become  secondarily 
infected  and  have  undergone  suppuration  the  eosinophilia 
disappears.  An  eosinophilia  is  not  invariable  in  these 
parasitic  infections,  and  may  be  absent  in  uncomplicated 
cases.  As  an  aid  to  diagnosis  in  doubtful  cases,  the  presence 
of  a  well-marked  eosinophilia  is  of  value;  a  negative  blood 
examination  is  of  less  significance. 

Skin  lesions. — Eosinophilia  is  a  condition  associated  with 
many  varieties  of  affections  of  the  skin.  It  is  present  in 
urticaria,  in  psoriasis,  and  in  dermatitis  herpetiformis.  In 
the  latter  condition  the  majority  of  the  cells  present  in  the 
bullae  may  be  eosinophils. 

In  the  specific  fevers  associated  with  skin  eruptions,  and 
already  mentioned  as  being  accompanied  by  inflammatory 
changes  in  the  blood,  a  considerable  eosinophilia  is  present  in 
the  early  stages.  In  small-pox  the  number  of  eosinophils 
may  be  very  high ;  in  chicken-pox  and  in  scarlet-fever  the 
condition  is  not  so  marked.  Both  in  small-pox  and  in  chicken- 
pox  eosinophils  are  also  present  in  the  vesicles,  but  disappear 
from  the  blood  and  from  the  skin  lesions  when  suppuration 
takes  place. 

Blood  diseases. — As  already  stated,  the  eosinophils  are 
increased  in  myeloid  leukaemia  and  to  a  less  extent  in  per- 
nicious anaemia.  Following  excision  of  the  spleen  in  animals 
Ehrlich  found  the  eosinophils  increased  after  a  considerable 
period;  a  similar  result  has  been  noted  to  occur  in  human 
beings,  but  as  a  rule  the  relative  increase  in  these  cells  is 
very  slight. 

Spasmodic  asthma  is  accompanied  by  an  increase  in  the 
eosinophils  of  the  blood  and  of  the  sputum. 


THE    SECONDARY   BLOOD   CHANGES.  21 

Spring  catarrh,  a  rare  affection  of  the  eyes,  is  associated 
with  an  eosinophilia  in  the  blood  and  with  large  numbers  of 
eosinophil  cells  in  the  conjunctival  discharge. 

Gonorrhoea  is  accompanied  by  a  slight  eosinophilia  in 
adults.  In  children  I  have  seen  an  eosinophilia  of  40  per 
cent,  in  a  case  of  gonorrhceal  affection  of  the  eye.  There  was 
no  evidence  of  intestinal  parasites  in  this  case.  Occasional 
eosinophils  are  seen  as  a  rule  in  films  made  from  the  pus  of  a 
gonorrhceal  discharge. 

(3)  Conditions  associated  with  a  leucopenia. 

Chronic  inflammation. — The  blood  changes  associated 
with  the  chronic  infective  granulomata,  tuberculosis,  and 
syphilis  are  the  reverse  of  those  set  up  by  the  pyogenic 
organisms. 

The  total  number  of  leucocytes  is  diminished  usually  to 
between  3  and  4  thousand  per  c.mm.,  and  the  relative  number 
of  the  lymphocytes  is  increased.  The  type  of  lymphocyte 
usually  increased  is  the  small  lymphocyte,  a  cell  identical 
with,  or  at  any  rate  very  similar  to,  the  lymphoid  cell  found 
in  the  giant  cell  system  of  tuberculosis  and  in  the  serous 
effusions  associated  with  tuberculous  and  syphilitic  diseases. 
In  addition  to  the  leucocytic  changes  there  may  be  a  consider- 
able reduction  in  the  number  of  red  cells  and  in  the  percentage 
of  haemoglobin.     The  colour  index  is  low. 

A  typical  blood  examination  would  give  the  following 
result : — 


Red  cells 
Haemoglobin 
Colour  index 

3,500,000  per  c.mm. 
45  per  cent. 
0-6 

White  cells    . 
Differential  count — 

3,000  per  c.mm. 

Polynuclear  neutrophils 
Eosinophils 
Large  hyalines 
Small  lymphocytes 
Large  lymphocytes 

40  per  cent. 

3 

4 
40 
13 

100 

In  acute   general    tuberculosis,  and    particularly    in    wide- 
spread   tuberculous    lymphadenitis,    the    blood    changes   are 


22  CLINICAL   PATHOLOGY. 

frequently  those  of  acute  inflammation,  as  has  been  mentioned 
under  that  heading. 

Fevers. — Certain  fevers  are  associated  with  similar  blood 
changes,  the  more  important  being  typhoid  fever,  influenza, 
malaria,  and  measles.  The  predominating  lymphocyte  in 
typhoid  and  in  malaria  may  be  of  the  large  type.  In  a 
febrile  case  of  doubtful  nature  a  complete  white  cell 
examination  may  be  of  considerable  assistance  in  diagnosing 
between  the  acute  inflammatory  and  the  non-suppurative 
affections,  as,  for  example,  between  a  pneumococcal  infection 
and  influenza. 

Pseudo-blood  diseases. — By  these  are  meant  certain 
diseases  of  unknown  etiology  which  affect  the  hsemopoietic 
system  and  which  do  not  give  rise  to  any  characteristic 
changes  in  the  blood,  other  than  the  secondary  anaemia 
associated  with  a  leucopenia  and  a  relative  lymphocytosis 
common  to  so  many  conditions.  These  diseases  include 
Hodgkin's  disease,  or  lymphadenoma,  splenic  anaemia,  and 
Banti's  disease.  In  none  of  these  affections  can  a  diagnosis 
be  made  from  the  results  of  a  blood  examination  alone. 

In  Hodgkin's  disease  the  general  glandular  enlargement, 
together  with  the  increase  in  the  size  of  the  spleen,  may  lead 
to  a  diagnosis  of  lymphoid  leukaemia,  and  this  can  be  negatived 
at  once  by  a  blood  examination.  The  two  conditions,  how- 
ever, have  less  clinical  similarity  than  might  be  expected. 
In  lympho- sarcoma  and  in  glandular  tuberculosis  there  is 
no  constant  blood  change  capable  of  distinguishing  the 
glandular  enlargements  from  those  of  Hodgkin's  disease.  A 
large  number  of  nucleated  red  cells  with  the  appearance  of 
myeloid  leucocytes  would  be  in  favour  of  sarcomatous  or 
carcinomatous  glands  with  other  deposits  in  the  bone  marrow. 
The  presence  of  a  high  polynuclear  leucocytosis  would  be  in 
favour  of  tuberculous  disease.  A  moderate  eosinophilia  is 
not  uncommon  in  Hodgkin's  disease.  The  only  certain 
method  of  diagnosis,  however,  consists  in  the  removal  of  a 
gland  for  histological  examination,  a  proceeding  which  in  the 
case  of  one  of  the  discrete  and  superficial  glands  may  readily 
be  carried  out  under  local  anaesthesia. 

Splenic  anaemia  is,  on  clinical  grounds  alone,  difficult  to 
distinguish  from  myeloid  leukaemia,  but  the  latter  disease  can 
be  recognised  at  once  by  a  blood  examination.     In   splenic 


THE    SECONDARY   BLOOD   CHANGES.  23 

anaemia  the  red  cell  and  haemoglobin  loss  may  not  be  great, 
but  the  diminution  in  the  number  of  leucocytes  is  more 
marked  than  in  any  other  condition.  The  white  cells  may 
fall  below  1,000  per  c.mni.  and  are  commonly  between  2,000 
and  3,000  per  c.mm.  Such  a  blood  condition  associated  with 
great  enlargement  of  the  spleen  is  scarcely  seen  in  any  other 
disease,  with  the  possible  exceptions  of  kala-azar  and  malaria. 

Banti's  disease  differs  from  splenic  anemia  in  the 
additional  association  of  cirrhosis  of  the  liver,  together  with 
haematemesis,  jaundice,  and  ascites  in  the  later  stages. 
Banti's  disease  is  usually  met  with  in  young  adults,  splenic 
anaemia  in  older  people  ;  but  the  two  diseases  are  not  clearly 
differentiated,  and  the  two  terms  may  apply  to  two  stages 
of  the  same  disease  or  may  embrace  a  variety  of  different 
affections.     The  blood  changes  are  identical. 

In  addition  to  the  diseases  already  enumerated,  this  type 
of  leucocytic  change  is  also  met  with  in  numerous  conditions 
which  affect  more  noticeably  the  red  cells  and  haemoglobin. 
A  leucopenia  with  a  relative  lymphocytosis  has  already  been 
stated  to  occur  in  chlorosis  and  pernicious  anaemia  among 
the  primary  blood  diseases  ;  they  are  also  found  in  the 
anaemias  of  malignant  growths,  among  workers  in  metallic 
poisons,  and  among  the  subjects  of  malignant  malaria  and 
other  tropical  diseases. 

(4)  Conditions  associated  with  an  increase  in  the  number 
of  red  cells  (polycythaemia  or  erythraemia). 

It  must  be  remembered  that  the  normal  number  of  red  cells 
in  a  healthy  male  adult  is  commonly  about  6  million  per  c.mm. 
and  that  any  number  in  the  neighbourhood  of  this  figure  does 
not  constitute  polycythaemia.  The  red  cell  estimations  quoted 
in  some  text-books  as  examples  of  polycythaemia  are  practically 
those  of  normal  persons. 

The  number  of  red  cells  is  capable  of  accommodation  to 
circumstances,  and  is  increased  at  high  altitudes,  during 
starvation,  and  temporarily  after  the  removal  of  large 
quantities  of  fluid  from  the  body,  as  after  tapping  an  ascitic 
abdomen.  Polycythaemia  is  accompanied  by  an  increase  in 
the  haemoglobin  percentage,  and  is  found  in  the  following 
morbid  conditions  : — 

Cardiac   failure,   accompanied  by  cyanosis   and   general 


24  CLINICAL '  PATHOLOGY. 

venous  stasis,  leads  to  an  increase  in  the  number  of  red  cells 
in  the  peripheral  circulation.  The  number  frequently  varies 
between  7  and  8  million  per  c.rnm. 

Congenital  morbus  cordis  is  almost  invariably  accom- 
panied by  a  considerable  polycythemia,  the  number  of  red 
cells  being  commonly  above  8  million.  Polycythemia  may 
be  present  when  the  cyanosis  is  by  no  means  marked ;  it  is 
temporarily  reduced  by  bleeding. 

Splenic  polycythsemia  (Erythremia  :  Osier's  disease  : 
Vaquez's  disease)  is  conveniently  considered  here,  although 
the  marked  alterations  in  the  blood  and  our  complete  ignorance 
as  to  the  cause  of  the  disease  would  justify  its  classifica- 
tion among  the  primary  blood  diseases.  It  is  a  comparatively 
rare  affection.  The  condition  is  a  chronic  one,  attacking 
people  usually  of  middle  age,  and  the  most  noticeable  clinical 
features  in  an  advanced  and  typical  case  are  the  striking 
plum-coloured  complexion  and  the  great  enlargement  of  the 
spleen. 

The  blood  obtained  on  puncturing  the  ear  is  almost  black 
in  colour,  and  so  sticky  that  it  is  difficult  to  obtain  sufficient 
for  a  count  without  pressure  and  impossible  to  spread  tbin 
films  of  it.  If  the  blood  is  taken  into  a  test  tube  and  centri- 
fuged  the  red  cells  are  found  to  almost  fill  the  serum,  leaving 
only  a  small  layer  of  supernatant  fluid.  The  actual  volume 
of  blood  is  greatly  increased.  The  number  of  red  cells  is 
commonly  about  10  million  per  c.mm.,  and  may  reach 
12  million.  The  percentage  of  hemoglobin  may  be  130  or 
over.  The  leucocytes  are  increased  to  about  double  the 
normal  number,  and  there  may  be  some  relative  increase  in 
the  poly  nuclear  neutrophils. 

(5)  Conditions  associated  with  a  decrease  in  the  number  of 
red  cells  (oligocythemia). 

A  decrease  in  the  number  of  red  cells  is  invariably  associated 
with  a  decrease  in  the  percentage  of  hemoglobin,  and  it  is 
this  blood  condition  which  is  commonly  referred  to  by  the 
loose  clinical  expression  "anemia."  If  the  unqualified  term 
"  anemia  "  is  used  at  all  it  must  be  applied  to  designate  a 
physical  sign  and  never  as  the  diagnosis  of  a  disease. 

The  clinical  recognition  of  the  anemic  state  is  apparently 
impossible.    Nothing  can  be  more  fallacious  than  the  common 


THE    SECONDARY  BLOOD   CHANGES.  25 

idea  that  the  colour  of  a  patient's  face,  or  even  of  his  lips 
and  conjunctivae,  is  any  guide  to  the  extent  of  his  anaemia. 
I  have  known  a  sallow-complexioned  constipated  Hebrew  with 
6  million  red  cells  per  c.mm.  and  105  per  cent,  of  haemoglobin 
diagnosed  as  pernicious  anaemia,  and  I  have  frequently  fallen 
into  the  error  of  expecting  in  a  patient  a  considerable  reduction 
in  the  red  cells  and  have  found  little  or  none. 

The  erythrocytic  mechanism  of  an  adult  in  ordinary  health 
is  very  evenly  regulated,  and  any  serious  drop  in  the  red  cells 
or  in  the  haemoglobin  content  is  to  be  regarded  as  a  definite 
indication  of  organic  disease. 

The  following  are  among  the  more  important  conditions 
associated  with  a  diminution  in  the  red  cells  and  haemoglobin. 

Haemorrhage  is  the  simplest  and  most  obvious  cause  of 
blood  loss,  and  is  naturally  followed  at  once  by  a  proportionate 
loss  in  red  cells  and  haemoglobin.  The  red  cells  formed  with 
great  rapidity  to  replace  those  lost  are  deficient  in  haemoglobin, 
consequently  the  colour  index  quickly  falls.  After  a  single 
haemorrhage  the  blood  readily  returns  to  normal,  in  periods 
varying  with  the  amount  of  blood  lost  and  the  recuperative 
powers  of  the  individual.  The  blood  lost  by  a  normal  person 
during  an  operation  attended  with  considerable  haemorrhage 
should  be  replaced  in  from  2  to  3  weeks.  Repeated 
small  haemorrhages  may  lead  to  a  considerable  degree  of 
anaemia,  and  if  the  bleeding  has  escaped  observation  the 
condition  of  the  patient  may  come  to  resemble  that  of 
pernicious  anaemia.  The  following  may  be  given  as  an 
example  of  the  condition  in  such  a  case. 

Fresh  blood. — Rouleaux  formation  slight.  Fibrin  forma- 
tion normal  or  excessive.     Poikilocytosis  present : — 

Red  cells         2,500,000  per  c.mm. 

Haemoglobin 30       per  cent. 

Colour  index  .. .         ...         ...  0*6        ,, 

White  cells  normal  in  number  or  increased. 

Stained  blood. — Polychromatophilia  present,  but  not 
extreme.  Occasional  normoblasts  seen,  but  no  megaloblasts. 
White  cells  often  show  some  increase  in  the  relative  numbers 
of  polynuclear  neutrophils. 

Such  a  blood  condition  is  found  not  only  after  haemorrhage, 
but  in  numerous  conditions  associated  with  "  anaemia,"  and  is 
of  the  kind  referred  to  as  of  the  chlorosis  or  of  the  secondary 


26  CLINICAL   PATHOLOGY. 

anaemia  type.  The  state  differs  from  that  of  chlorosis  in  that 
the  red  cells  are  commonly  more  affected  and  the  colour  index 
of  necessity  higher,  though  these  differences  may  not  be 
marked,  and  are  of  less  consequence  since  the  clinical  recogni- 
tion of  the  disease  chlorosis  is  rarely  difficult.  It  is  of  great 
importance  to  distinguish  this  secondary  type  of  anaemia 
from  primary  or  pernicious  anaemia.  The  main  differences 
in  the  secondary  type  are  the  rarity  with  which  the  red  cells 
fall  below  2  million,  a  colour  index  less  than  0*8,  and  the 
absence  of  megaloblasts. 

Metallic  poisons. — Workers  in  arsenic,  antimony,  lead 
and  similar  metals  may  develop  an  anaemia  of  the  secondary 
type.  Basic  granular  degeneration  of  the  red  cells  is  said 
to  be  especially  characteristic  of  lead  poisoning.  Granular 
degeneration  is  present  rarely  in  other  diseases  accompanied 
by  a  secondary  anaemia,  and  is  seen  in  its  greatest  degree  in 
pernicious  anaemia.  In  cases  of  lead  poisoning  admitted  to 
a  general  hospital  I  have  hardly  ever  seen  marked  granular 
degeneration  of  the  red  cells.  On  the  Continent,  however,  the 
presence  and  relative  proportion  of  these  cells  is  taken  as 
evidence  of  the  extent  to  which  the  lead- worker  is  affected. 
The  Cachexias  of  carcinoma,  tuberculosis,  and  syphilis  are 
associated  with  this  type  of  anaemia,  and  when  the  physical 
signs  of  these  conditions  are  latent  the  discovery  of  serious  loss 
in  red  cells  and  haemoglobin  is  sufficient  indication  for  further 
investigations.  Gout,  diabetes,  myxoedema,  and  Addison's 
disease  are  among  the  numerous  chronic  affections  accompanied 
by  erythrocytic  changes.  In  addition,  diseases  associated  with 
blood  changes  more  especially  affecting  the  leucocytes,  and 
which  have  been  already  mentioned,  are  usually  accompanied 
by  red  cell  changes  in  addition.  The  anaemias  due  to  intestinal 
parasites  may  be  particularly  severe. 

The  Blood  Changes  occueeing  in  Childeen. 

Before  considering  the  changes  which  may  be  found  in  the 
blood  of  infants  it  is  necessary  to  appreciate  that  in  children 
less  than  5  years  old  the  normal  blood  presents  several 
differences  from  that  of  adults  and  tends  to  react  excessively  to 
abnormal  stimuli. 

In   infants    the   total    leucocyte   count    is   high   and    the 


THE    SECONDARY    BLOOD   CHANGES.  27 

lymphocytes  relatively  numerous.  Nucleated  red  cells  are 
present  at  birth,  and  persist  for  several  months  after  birth. 
The  blood  readily  reverts  to  the  foetal  type  in  disease,  and 
remarkable  fluctuations  in  the  number  and  character  of  the 
cells  such  as  would  indicate  the  gravest  disorders  in  an  adult 
may  have  little  serious  meaning  in  an  infant.  The  blood- 
forming  mechanism  in  an  infant  has  had  much  the  same  time 
to  steady  down  as  its  heat-regulating  centre,  and  both  may  be 
temporarily  disordered  by  the  cutting  of  a  tooth. 

The  extreme  characters  of  the  fluctuations  in  the  blood  of 
infants  renders  the  pathology  of  the  blood  very  obscure  and 
any  classification  of  the  blood  diseases  of  children  almost 
impossible.  The  matter  is  further  complicated  by  the 
comparative  frequency  with  which  the  infantile  spleen  and 
lymph  glands  enlarge. 

Lymphoid  leukaemia,  Myeloid  leukaemia,  and 
Pernicious  anaemia  have  all  been  described  as  occurring  in 
infants,  and  it  is  possible  that  a  very  few  of  the  reported 
cases  are  genuine.  Such  diagnoses  should  be  made  with  the 
utmost  reserve  and  never  on  the  examination  of  a  blood 
film  alone.  If  that  is  to  be  taken  as  the  criterion  of  our 
diagnosis,  then  I  have  seen  an  infant  pass  calmly  through  an 
attack  of  acute  lymphoid  leukaemia  with  nothing  more  deadly 
than  a  whoop  and  another  child  shake  off  in  rapid  succession 
the  onslaughts  of  myeloid  leukaemia  and  pernicious  anaemia. 
The  blood  changes  of  these  diseases  are  better  regarded  as 
types  of  the  changes  which  may  accompany  some  of  the 
known  affections  of  childhood. 

The  blood  changes  of  infants  may  be  divided  into  : — 

(1)  The  primary  blood  diseases  of  infants. 

(2)  The  secondary  blood  diseases  of  infants. 

(1)  The  primary  blood  diseases  of  infants. 

Splenic  anaemia  of  infants  (Von  Jaksch's  anaemia : 
Anaemia  infantum  pseudoleukaemica) . — This  is  a  disease 
affecting  quite  young  children  and  accompanied  by  great 
enlargement  of  the  spleen  and  moderate  enlargement  of  the 
liver.  The  affected  child  may  recover  completely,  or  may  die, 
or  may  improve  and  be  left  with  a  large  spleen  and  subse- 
quently come  into  the  category  of  Banti's  disease.  It  is  still 
a  matter  of  dispute  as  to  whether  the  disease  constitutes  a 


28  CLINICAL  jPATHOLOGY. 

clinical  entity  or  is  a  condition  secondary  to  rickets  or 
congenital  syphilis.  Undoubted  cases  are,  however,  met  with 
in  which  no  evidence  of  either  disease  can  be  found  and  in 
which  the  Wasserrnann  reaction  is  negative.  Such  cases 
present  a  fairly  typical  clinical  picture  and  a  remarkable,  if 
somewhat  varied,  blood  condition,  so  that  it  is  reasonable  to 
describe  them  for  the  present  as  examples  of  the  primary 
anaemias  of  children. 

The  main  features  of  the  blood  condition  are  as  follow: — 

The  red  cells  and  haemoglobin  percentage  are  greatly 
reduced,  the  former  to  a  number  between  1  and  2  million 
per  c.mm.,  the  latter  to  from  10  to  30  per  cent. 

The  white  cells  are  increased  often  to  a  marked  degree,  and 
frequently  number  from  30  to  40,000  per  c.mm. 

The  stained  film  is  very  remarkable,  and  may  display  every 
known  kind  of  blood  cell  in  considerable  numbers.  Nucleated 
red  cells  are  always  present,  and  usually  in  excessive  numbers ; 
200  may  be  seen  while  counting  500  leucocytes.  Normoblasts 
are  greatly  in  excess  of  megalo blasts.  The  relative  percentage 
of  the  leucocytes  is  very  variable,  and  the  predominant  cells 
may  be  lymphocytic,  but  are  more  frequently  myeloid. 
Myelocytes,  especially  the  small  type  of  neutrophilic  myelo- 
cyte, are  numerous. 

More  rarely  a  great  enlargement  of  the  spleen  may  be 
associated  with  extreme  red  cell  changes,  a  high  colour  index, 
and  little  or  no  change  in  the  leucocytes. 

Congenital  family  cholsBmia  (Acholuric  family 
jaundice)  is  a  rare  and  interesting  hereditary  condition 
occurring  in  several  members  of  the  same  family,  and 
associated  with  enlargement  of  the  spleen,  the  presence  of  bile 
in  the  serum,  and  the  usual  absence  of  bile  from  the  urine.  The 
blood  in  the  course  of  an  ordinary  examination  shows  nothing 
more  than  a  moderate  secondary  anaemia  and  the  presence 
of  occasional  nucleated  red  cells.  The  erythrocytes  are  in 
reality  remarkable  by  reason  of  their  abnormal  fragility.  This 
fragility  is  demonstrated  by  the  ease  with  which  the  cells  are 
haemolysed  in  hypotonic  salt  solutions.  Normal  red  cells  retain 
their  haemoglobin  when  placed  in  solutions  of  sodium  chloride  of 
0*4  per  cent,  or  less ;  the  red  cells  in  this  disease  are  haeruolysed 
in  salt  solutions  approaching  the  strength  of  normal  saline. 

Haemophilia  is  a  comparatively  rare  hereditary  disease 


THE    SECONDARY   BLOOD   CHANGES.  29 

transmitted  by  unaffected  females  to  males  and  characterised 
by  extensive  haemorrhages  following  trifling  causes.  Affected 
children  rarely  reach  the  age  of  maturity.  Haemophilia  is 
commonly  classified  among  the  purpuras,  with  which  it  has 
probably  nothing  in  common.  The  essential  known  morbid 
change  is  the  alteration  in  the  coagulation  time  of  the  blood. 
The  normal  coagulation  time  of  the  blood  as  estimated  by 
Wright's  method  (page  46)  is  very  constant,  and  is  almost 
invariably  in  the  near  neighbourhood  of  3|  minutes.  In 
haemophilia  the  time  is  greatly  prolonged,  sometimes  to  as  much 
as  half  an  hour.  The  coagulation  time  diminishes  after 
severe  haemorrhage  or  after  calcium  salts  have  been  given  by 
the  mouth.  Hemophilics  can  be  distinguished  from  other 
patients  who  may  be  the  subjects  of  excessive  haemorrhage  by 
this  marked  retardation  in  the  coagulation  of  their  blood.  The 
ordinary  blood  examination  in  haemophilia  shows  nothing 
characteristic,  and  the  red  cells  are  not  excessively  fragile,  as 
in  acholuric  family  jaundice. 

(2)  The  secondary  blood  diseases  of  infants. 

Rickets  and  Congenital  syphilis  are  the  two  most 
important  primary  conditions  capable  of  producing  marked 
alteration  in  the  blood  of  children.  A  considerable  enlarge- 
ment of  the  spleen  and  liver  is  frequently  associated  and  a 
condition  produced  which  may  be  very  similar  to  that  of 
splenic  anaemia  infantum.  The  degree  of  anaemia  may  be 
marked  and  nucleated  red  cells  numerous.  In  some  cases 
the  leucocytes  are  greatly  increased,  in  others  there  may  be  a 
leucopenia,  with  a  relative  lymphocytosis.  The  blood  changes 
are  very  varied  and  rarely  present  to  the  degree  seen  in 
splenic  anaemia,  from  which  the  secondary  blood  diseases  are 
to  be  diagnosed,  partly  by  the  minor  enlargement  of  the 
spleen  and  the  less  obvious  blood  alteration,  but  mainly  by  a 
recognition  of  the  originating  factor.  A  relative  lymphocytosis 
is  common  in  many  affections  of  childhood,  including  tuber- 
culosis, and  more  especially  whooping  cough.  In  these 
diseases  there  may  be  a  marked  increase  in  the  total  leucocytes 
and  a  blood  picture  produced  very  similar  to  that  of  lymphatic 
leukaemia.  The  lymphocytes  present  are  usually  identical 
with  the  small  lymphocytes  of  normal  blood,  and  have  a 
deeply-staining  round  nucleus. 


30  CLINICAL   PATHOLOGY. 

Infantile  scurvy  (Barlow's  disease)  is  usually  associated 
with  a  severe  anaemia  of  the  secondary  type  and  as  a  rule  with 
an  inflammatory  leucocytosis. 

Purpura  in  its  various  forms  is  not  confined  to  children, 
but  occurs  in  them  more  commonly  than  in  adults.  The  most 
extensive  purpura  is  not  accomj)anied  by  any  characteristic 
changes  in  the  blood.  As  a  rule  there  is  a  mild  secondary 
anaemia  and  the  leucocytes  are  unaltered.  The  coagulation 
time  is  normal,  and  there  is  no  increased  fragility  of  the  red 
cells.  The  serum  usually  has  the  property  of  agglutinating 
the  red  cells  of  unaffected  individuals.  If  the  serum  of  a 
purpuric  individual  is  added  to  the  washed  red  cells  of  a 
normal  person  the  red  cells  are  thrown  into  tight  clumps 
similar  to  the  clumps  of  typhoid  bacilli  in  a  positive  Widal 
reaction.  The  purpuric  red  cells  are  not  agglutinated  by  the 
serum  of  a  similar  case.  Hemagglutinins  in  the  serum  are 
of  scientific  rather  than  of  practical  interest,  and  red  cell 
agglutination  of  the  nature  usually  found  in  purpura  may  be 
present  in  a  variety  of  conditions  and  occasionally  in  apparently 
healthy  individuals  (page  58). 

Summary. 

The  secondary  blood  changes  of  adults  may  be  divided 
into  those  which  mainly  affect  the  leucocytes  on  the  one 
hand  and  the  red  cells  on  the  other.  The  leucocytic  changes 
are  of  two  main  types,  namely,  the  leucocytosis,  usually 
polynuclear  in  character,  set  up  by  the  pyogenic  organisms, 
and  the  leucopenia,  with  a  relative  lymphocytosis  which 
accompanies  the  non-suppurative  infections. 

To  these  must  be  added  the  small  group  of  disorders 
associated  with  an  eosinophilia. 

The  erythrocytic  changes  are  similarly  two.  An  increase  in 
red  cells  and  haemoglobin  is  comparatively  rare  ;  a  decrease 
is  met  with  in  the  great  majority  of  serious  and  prolonged 
diseases,  and  is  always  an  indication  of  notable  organic 
disturbance. 

The  fluctuations  of  the  blood-forming  mechanism  in  infants 
are  difficult  to  classify  and  to  interpret  owing  to  the  relatively 
foetal  condition  of  the  infantile  blood  in  health  and  to  the 
excessive  response  of  the  mechanism  to  morbid  stimuli. 

The  main  changes  of  the  primary  blood  diseases  of  adults 


THE    SECONDARY   BLOOD   CHANGES.  31 

may  temporarily  appear  in  infants,  the  diseases  themselves 
very  rarely  indeed.  The  condition  known  as  splenic  anasmia 
infantum  appears  to  be  a  clinical  entity,  and  may  be  regarded 
as  such  in  spite  of  the  fact  that  very  similar  blood  changes 
may  be  set  up  by  other  conditions,  and  particularly  by  rickets 
or  congenital  syphilis. 


CHAPTEE  III. 

THE    METHODS    OF    EXAMINING    THE    BLOOD. 

The  ordinary  routine  examination  of  the  blood  includes  the 
following  investigations  : — 

(1)  The  unstained  blood. 

(2)  The  percentage  of  haemoglobin. 

(3)  The  estimation  of  the  number  of  red  cells  to  the  cubic 

millimetre. 

(4)  The  estimation  of  the  number  of   leucocytes  to  the 

cubic  millimetre. 

(5)  The  stained  blood. 

The  essential  apparatus  comprises  : — 

A  microscope. 

A  haenioglobinometer. 

Blood  pipettes  and  diluting  fluid. 

A  haemocytometer. 

A  special  blood  stain. 

Slides  and  cover  glasses. 

A  dry  swab,  ether,  and  a  surgical  needle. 
The  majority  of  these  materials  are  described  under  their 
appropriate    headings.       The     microscope     is     conveniently 
referred   to   here. 

The  microscope. — A  thoroughly  reliable  instrument  is 
necessary,  not  only  for  the  examination  of  the  blood,  but  for 
numerous  other  pathological  investigations  in  common  use. 
For  a  student  who  is  not  hampered  b}^  motives  of  economy  or 
by  the  previous  possession  of  an  inferior  microscope  the 
choice  is  not  difficult,  if  he  bears  in  mind  the  following  points. 
The  microscope  should  have  a  firm  base,  should  be  provided 
with  a  diaphragm  and  Abbe  condenser,  with  a  mechanical 
stage,  with  a  triple  nose-piece  for  objectives  having  the  focal 
distances  of  f ,  -jt,  and  ^  inch,  and  with  at  most  three  eye- 
pieces No.  1  (X  4  diameters),  No.  2  (X  6  diameters),  No.  4 
( X  10  diameters).  The  low-power  eye-piece  should  be  used 
for  all  ordinary  work.     The  mechanical  stage  should  be  built 


THE    METHODS   OE   EXAMINING   THE   BLOOD.    33 


into  and  form  part  of  the  framework  of  the  microscope  ;  it 
need  not  necessarily  be  graduated.  Thoroughly  reliable 
microscopes  are  manufactured  by  several  of  the  leading 
English  firms,  and  there  is  no  necessity  to  obtain  an  imported 
instrument.  The  ^  inch  objectives  of  home  manufacture 
however,  are  often  inferior  to  those  made  by  Zeiss,  and  the 
additional  cost  of  the 
Zeiss  lens  is  worth  the 
outlay ;  a  Zeiss  J  inch 
objective  is  a  further 
but  not  essential  advan- 
tage. The  best  English 
microscope  fitted  with  a 
Zeiss  TX2  inch  objective 
costs  about  £21. 

If  necessary  the  triple 
nose-piece  can  be  dis- 
pensed with  and  a  double 
or  even  single  nose- 
piece  used.  A  mechani- 
cal stage  can  be  fitted  to 
almost  any  microscope, 
but  is  seldom  entirely 
satisfactory,  since  the 
numerous  patterns  in 
use  require  constant 
attention  and  easily  get 
out  of  order.  The  stage 
is  a  great  convenience, 
but  is  not  absolutely 
essential,  unless  Strong's  method  of  counting  the  leucocytes 
is  used. 

To  obtain  the  blood. — Rub  the  lobe  of  the  ear  lightly 
with  ether  and  allow  it  to  dry.  With  as  rapid  a  movement  as 
possible  make  a  deep  puncture  with  a  surgical  needle.  The 
novice  is  apt  to  deliberately  press  the  needle  into  the  ear, 
giving  the  maximum  amount  of  pain  and  obtaining  the 
minimal  quantity  of  blood.  By  a  rapid  stab  sufficient  blood 
can  be  obtained  from  a  sleeping  infant  without  awakening  it. 
The  best  needle  to  employ  is  the  ordinary  straight  Hagedorn 
No.  9.      It  should  be  sterilised  before  use  by  rapid  passage 

p.  3 


Fig.  1.— The  Microscope. 


34  CLINICAL   PATHOLOGY. 

several  times  through  a  flame.  The  specialised  instruments 
of  torture  provided  with  nearly  all  forms  of  blood  apparatus 
should  be  avoided.  The  drop  of  blood  must  be  allowed  to  flow 
out,  and  should  never  be  squeezed  out,  since  anjr  pressure 
upsets  the  equilibrium  between  cells  and  plasma.  Sufficient 
blood  can  rarely  be  got  from  the  finger  without  considerable 
pressure  ;  it  can  always  be  obtained  from  the  ear. 

The  unstained  blood. — Place  a  cover-glass  with  one  edge 
flush  with  the  edge  of  a  slide.  Hold  the  two  lightly  in  apposi- 
tion with  the  thumb.  Place  the  apposed  edges  against  a  drop 
of  blood  and  the  blood  will  flow  between  slide  and  cover-glass. 
Examine  within  10  minutes  or  so,  using  the  J  inch  objec- 
tive. All  the  available  light  should  be  thrown  through  the 
condenser,  and  the  diaphragm  should  then  be  closed  down 
until  the  corpuscles  stand  out  clearly.  If  the  diaphragm  is 
left  widely  open  the  cells  cannot  be  seen  at  all. 

Observe  the  extent  of  rouleaux  and  fibrin  formation,  the 
collections  of  blood  platelets,  the  shape  and  size  of  the  red 
cells,  and  the  relative  proportion  of  white  to  red  cells.  Gross 
changes  in  the  blood  such  as  occur  in  the  leukaemias  can  be 
recognised  at  once  by  this  simple  process  ;  some  of  the  minor 
changes  can  be  observed  in  no  other  way,  and  it  should  never 
be  omitted.  Spirilla,  filaria,  and  trypanosomes  can  be 
readily  recognised  if  present.  Malarial  parasites  are  more 
certainly  identified  with  a  j1^  inch  objective ;  the  eye  is 
attracted  to  them  by  the  active  dancing  of  their  pigment 
granules. 

The  hemoglobin. —  Some  form  of  apparatus  is  necessary 
for  the  estimation  of  haemoglobin,  the  blotting  paper  method 
of  Tallqvist  is  too  unreliable  for  anything  more  than  an 
approximate  reading. 

Haldane's  modification  of  Gower's  instrument  is  the 
most  convenient  hsemoglobinometer  for  general  use.  The 
standard  of  comparison  in  this  form  of  hsemoglobinonieter 
consists  of  a  tube  containing  a  1  per  cent,  solution  of  blood 
having  the  average  percentage  of  haemoglobin  found  in  the 
blood  of  a  healthy  adult  man,  and  which  has  been  saturated 
with  carbonic  oxide.  A  measured  quantity  of  the  blood  to  be 
examined  is  placed  in  a  similar  graduated  tube,  saturated  with 
carbonic  oxide  and  diluted  until  it  matches  the  standard  tube 
in  colour.    The  readings  obtained  by  this  instrument  are  with 


THE   METHODS   OF  EXAMINING   THE   BLOOD      35 

practice  very  exact,  and  it  is  claimed  that  the  error  does  not 
exceed  1  per  cent. ;  it  should  not  exceed  5  per  cent.,  and  that 


Fig.  2. — The  Normal  Unstained  Blood. 


Fig.  3. — The  Unstained  Blood  in  Myeloid  Leukaemia. 

3—2 


36 


CLINICAL   PATHOLOGY. 


is  all  that  is  necessary  for  ordinary  purposes.  The  instruc- 
tions for  use  are  supplied  with  the  instrument  and  are  briefly 
as  follow :  — 

20  c.mm.  of  blood  are  drawn  up  into  the  pipette  and  the  end 
of  the  pipette  is  wiped.  A  small  quantity  of  distilled  water 
having  been  placed  in  the  graduated  tube  the  blood  is  blown 
into  this,  the  pipette  rinsed  up  and  down  with  the  water  and 
withdrawn.  A  piece  of  rubber  tubing  is  affixed  to  a  gas  jet  by 
one  end  and  the  other  end  is  passed  into  the  tube  to  near  the 
level  of  the  mixture  of  blood  and  water.     The  gas  is  allowed 


Pig.  4. — Haldane's  Hteraoglobinometer. 

to  pass  for  a  few  seconds,  the  rubber  tube  is  drawn  out  with 
the  gas  still  escaping,  the  end  of  the  graduated  tube  closed 
with  the  ringer,  and  the  tube  slowly  inverted  several  times. 
The  diluted  blood  saturated  with  carbonic  oxide  is  then  com- 
pared with  the  standard  tube  and  carefully  diluted  further 
until  the  colour  in  the  two  tubes  is  exactly  matched.  The 
level  of  the  diluted  liquid  gives  the  percentage  of  the  haemo- 
globin. In  matching  the  colour  the  tubes  should  be  held 
against  the  light  from  the  sky  and  frequently  transposed. 

Oliver's    haemoglobinometer  as  modified  and    manu- 
factured by  the  Tintometer  Co.,  Ltd.,  of  Salisbury,  is  a  more 


THE   METHODS   OF   EXAMINING   THE   BLOOD.    37 

expensive  and  somewhat  cumbersome  apparatus.  It  is,  how- 
ever, accurate,  simple  to  use,  and  does  not  require  a  supply  of 
coal  gas.  The  tintometer  consists  of  a  wooden  box  illuminated 
by  a  candle,  and  is  provided  with  a  small  capillary  pipette,  a 
mixing  chamber,  and  a  long  sliding  scale  containing  a  series 
of  tinted  discs  graduated  in  percentages. 
The  method  of  use  is  as  follows  : — 

Fill  the  capillary  pipette  by  holding  the  fine  end  against  a 
drop  of  blood.  No  suction  is  required.  Wipe  the  end  of  the 
pipette  and  slip  over  it  a  small  piece  of  rubber  tubing  attached 
to  an  ordinary  glass  pipette  rilled  with  distilled  water  and 
provided  with  a  rubber  teat.  Blow  out  the  blood  into  the 
mixing  chamber  together  with  sufficient  water  to  exactly  fill 
the  chamber.  Stir  the  mixture  with  the  metal  handle  of  the 
pipette  and  cover  the  chamber  with  the  piece  of  glass  provided. 
If  the  chamber  is  accurately  filled,  one  small  air  bubble  rests 
between  the  glass  and  the  drop  and  does  not  interfere  with 
the  observation.  Place  the  chamber  in  the  box,  light  the 
candle  and  shut  the  door  of  the  box.  Slide  the  graduated 
scale  into  the  box,  and,  shading  the  observing  eye  with  the 
hand  so  as  to  cut  off  all  external  light,  move  the  scale  in  and 
out  until  the  disc  is  found  which  exactly  matches  the  colour  of 
the  diluted  blood  in  the  chamber.  The  number  of  the  disc 
gives  the  percentage  of  haemoglobin  in  the  blood.  By  means  of 
the  graduated  rider  provided  to  fit  over  the  scale  the  percentage 
can  be  read  to  within  5  per  cent.  (Both  the  above-described 
instruments  are  provided  by  Messrs.  Hawksley,  of  Oxford  Street.) 
The  estimation  of  the  number  of  red  cells.— 
(1)  Strong's  method  (modified). — The  necessary  apparatus 
consists  of  two  graduated  pipettes,  a  mixing  bottle,  a  diluting 
fluid,  and  a  Thoma-Zeiss  hamiocytometer.  The  pipettes  are 
graduated  to  hold  995  c.mm.  and  5  c.mm.  respectively.  The 
5  c.mm.  tube  has  two  marks  close  together ;  from  the  upper 
mark  5  c.mm.  are  delivered,  at  the  lower  mark  they  are  con- 
tained. The  mixing  bottle  holds  just  over  1,000  c.mm.,  and 
is  provided  with  a  well-fitting  stopper. 

The  diluting  fluid  has  the  following  composition  : — 

•85  gramme  sodium  chloride. 

•85  gramme  sodium  citrate. 

Commercial  (40  per  cent.)  formalin  1  c.c. 

Distilled  water  to  100  c.c. 


38 


CLINICAL   PATHOLOGY. 


The  method  of  use  is  as  follows  : — 

995  c.mm.  of  diluting  fluid  are  measured  into  the  mixing- 
bottle  :  5  c.mm.  of  blood  are  drawn  up  to  the  lower  mark  in  the 
small  pipette.  The  end  of  the  tube  is  wiped,  placed  in  the 
bottle,  the  blood  blown  out,  and  the  pipette  rinsed  up  and 
down  with  the  mixture  of  blood  and  fluid.  The  mixing  bottle 
is  corked,  and  can  be  kept  until  it  is  convenient  to  count  the 
cells.  The  red  cells  sink  to  the  bottom,  and  before  counting 
the  bottle  must  be  thoroughly  shaken.     After  shaking,  a  drop 


Fig.  5. — Strong's  Pipettes  and  Mixing  Bottle. 

of  the  mixture  is  transferred  to  the  centre  disc  of  the  haerno- 
cytometer  by  means  of  the  stopper  or  a  glass  rod.  The  cover- 
glass  is  gently  lowered  on  to  the  drop  and  stroked  down  on 
the  haemocytometer  with  the  handles  of  two  mounted  needles 
until  circles  of  coloured  rings  (Newton's  rings)  appear  between 
slide  and  cover-glass.  The  rings  are  best  seen  by  holding 
the  slide  up  to  the  light  and  looking  down  at  it  in  a  slanting 
direction  from  a  little  distance  above.  The  size  of  the  drop 
when  flattened  out  in  this  manner  must  be  sufficiently  large 
to  nearly  fill  the  central  platform  of  the  counting  slide,  but 
not  so  large  that  any  of  it  flows  into  the  trench  surrounding 
the  platform.  No  air  bubbles  should  be  present  in  the  drop. 
The  slide  is  allowed  to  stand  for  a  few  minutes  while  the  cells 
settle  down,  and  is  then  placed  on  a  microscope  fitted  with 
a  No.  2  eye-piece  and  a  \  inch  objective.  The  light  is 
thrown  through   the  condenser,  and  the  diaphragm  is  shut 


THE   METHODS   OF   EXAMINING   THE   BLOOD.    39 


down  in  the  same  manner  as  when  examining  the  fresh  blood. 
The  microscope  must  be  vertical,  otherwise  the  red  cells  settle 
towards  the  lower  part  of  the  slide.  The  occasional  leucocytes 
which  may  be  present  are  readily  distinguished  from  the  red 
cells  by  their  shape,  colour,  and  the  usual  presence  of 
refractile  granules.  The  slide  is  moved  until  the  ruled  area 
of  the  central  disc  is  found.  This  area  is  ruled  in  small 
squares,  every  fifth  square  in  either  direction  having  a  line 
drawn  through  the  middle  of  it.  The  area  is  in  this  manner 
marked  off  into  16  large  squares,  each  of  which  contains 
16  small  squares  bounded  on  every  side  with  small  double- 
ruled  squares.  A  large  square  occupies  a  field  of  the 
microscope.  In  counting  the  red  cells  only  those  in  the 
single-ruled  squares  are  considered, 
and  as  a  minimum  all  the 
red  cells  in  4  large  squares,  each 
consisting  of  16  small  squares, 
are  counted.  In  cases  in  which 
the  red  cells  are  much  diminished 
all  the  16  large  squares  should  be 
counted.  A  certain  number  of  the 
red  cells  will  be  found  to  impinge 
on  the  lines  bounding  the  squares; 
such  cells  should  only  be  in- 
cluded when  lying  on  the  left- 
hand  and  bottom  lines  of  the 
squares.  The  average  number  of  red  cells  per  large  square 
in  health  is  about  120. 

In  order  to  calculate  the  number  of  red  cells  to  the  cubic 
millimetre  it  should  be  remembered  that  the  dimensions  of  a 
small  square  are  ^oo  of  a  cubic  millimetre  and  that  the  blood 
has  been  diluted  200  times.  The  number  of  red  cells  to  the 
cubic  millimetre  will  therefore  be  4000  X  200  X-  the  average 
number  of  red  cells  per  small  square.  If  400  red  cells  are 
counted  in  4  large  squares  containing  64  small  squares  the 
number  of  red  cells  to  the  c.mm.  of  undiluted  blood  would  be 

4,000  X  200  X  ^r°  or  5,000,000. 

(2)  The  Thoma-Zeiss  method. — The  apparatus  consists 
of  a  combined  pipette  and  mixing  chamber,  a  diluting  fluid, 
and  a  hsemocytometer.      The  diluting  fluid  employed  may  be 


,}-. «, '    ] 

■>  TT'TT^ 

*PT  °= 

T  7™t"t; 

Y  o  °  °  ;• 

:••"„:/!' 

.y.  :•  f : 

V  .%• 

te  {•. 

°i  y;  .v. 

"/."'•:"*. 

°'  °°° 

'T 

o    o     °  o         o 

v-  >l°i 

:  ' '«" " 

kvj: 

•f;.;.; 

°°  -  -- 

™ J  °  ° c 

Fig.  6.— Field  of  Hfemo- 
cytometer. 


40  CLINICAL   PATHOLOGY. 

the  same  as  that  described  under  Strong's  method,  or  a 
mixture  may  be  used  which  contains  a  stain  for  differen- 
tiating the  leucocytes.  The  staining  mixture  commonly 
employed  is  that  of  Toison,  and  has  the  following  com- 
position : — 

Methyl  violet  ....     0'25  gramme. 

Neutral  glycerine     ....        30  c.c. 

Distilled  water  ....        80  c.c. 

Add  to  this  a  solution  of  : — 

Sodium  chloride       .         .         .         .  1  gramme. 

Sodium  sulphate      ....  8  grammes. 

Distilled  water  ....         80  c.c. 

Filter  the  mixture. 

To  make  the  dilution  draw  up  the  blood  in  the  pipette 
to  the  mark  0'5.  Wipe  the  end  of  the  pipette.  Place  the 
pipette  in  a  bottle  of  the  diluting  fluid  and  draw  up  the 
fluid  to  the  mark  101.  Eotate  the  tube  vigorously  until 
blood  and  fluid  are  thoroughly  mixed  in  the  bulb  of  the 
pipette.  The  blood  in  the  mixing  bulb  is  now  in  a  dilution 
of  1  in  200.  Blow  out  the  fluid  in  the  capillary  part  of  the 
pipette,  also  a  few  drops  of  the  diluted  blood  in  the  bulb,  and 
transfer  a  drop  to  the  platform  of  the  counting  slide.  Proceed 
as  described  under  Strong's  method.  The  disadvantages 
of  this  method  are  that  it  is  difficult  to  be  sure  of  a  thorough 
mixing  of  the  blood  and  fluid  in  the  bulb,  and  that  the 
contents  of  the  pipette  tend  to  leak  out  and  necessitate 
the  immediate  counting  of  the  cells.  In  Strong's  method 
the  pipette  is  more  easily  manipulated,  the  mixture  is  readily 
transported  and  can  be  counted  at  leisure,  and  the  same 
mixture  can  be  used  without  further  apparatus  for  an 
enumeration  of  the  leucocytes. 

The  estimation  of  the  number  of  white  cells. — 
(1)  Strong's  method  (modified).— The  apparatus  required 
is  the  same  as  that  for  the  red  cells.  The  same  mixture  of 
5  c.mm.  of  blood  with  995  c.mm.  of  diluting  fluid  in  a  mixing 
bottle  is  employed.  After  thoroughly  shaking  the  bottle  draw 
up  the  mixture  of  blood  and  diluting  fluid  to  the  upper  of  the 
two  marks  on  the  5  c.mm.  pipette.  Wipe  the  end  of  the 
pipette.  Place  a  clean  slide  on  the  bench  and  hold  the  pipette 
vertically  to  the  slide  with  the  end  of  the  pipette  just  resting 
on  the  centre  of  the  slide.     Blow  out  the  fluid  in  the  form  of 


THE   METHODS   OF   EXAMINING    THE    BLOOD.    41 

a  drop,  lifting  the  pipette  and  ceasing  to  blow  just  as  the  last 
portion  of  the  fluid  falls  out.  Allow  the  drop  to  dry.  Filter 
hasniotoxylin  on  to  the  slide  for  5  minutes.  Wash  in  tap 
water  for  3  minutes.  Wipe  off  excess  of  water  and  allow 
the  drop  to  dry.  Do  not  blot  dry.  When  dry  mount  in  cedar 
wood  oil  with  a  cover  slip.  (Instead  of  hematoxylin,  Leishman's 
stain,  or  any  simple  nuclear  stain,  such  as  carbol  thionin,  may 
be  used.)  Place  in  the  eye-piece  of  the  microscope  a  flat 
round  metal  disc  with  a  central  square  aperture  f  of  an 
inch  square,  or,  failing  this,  make  a  square  hole  in  a  piece  of 
visiting  card  cut  to  the  size  of  the  eye- piece.  All  that  is 
required  is  to  obtain  a  square  field  for  counting  the  leucocytes 
in  a  round  drop.  Use  the  £  inch  objective.  Observe  that 
the  nuclei  of  the  leucocytes  are  stained  blue  and  the  red  cells 
are  practically  unstained.  The  edge  of  the  drop  is  clearly 
defined.  To  count  the  leucocytes  find  the  top  segment  of  the 
drop  and  move  the  drop  across  the  field  from  one  side 
to  the  other.  When  the  other  side  is  reached  mark  a 
red  cell  on  the  bottom  line  of  the  square  and  move  the 
drop  down  exactly  1  square  field.  Continue  moving  the 
drop  backwards  and  forwards  across  the  field  until  the  entire 
drop  has  been  covered  and  all  the  leucocytes  have  been 
counted.  The  number  of  white  cells  counted  in  a  normal 
case  would  be  about  150.  The  dilution  of  the  blood  is  1  in 
200,  and  5  c.mm.  of  this  dilution  have  been  counted.  The 
number  of  leucocytes  in  1  c.mm.  of  the  undiluted  blood  would 

therefore  be  150  multiplied  by  — — -,  or  6,000.     In  cases  of 

leukaemia  with  an  excessive  number  of  white  cells  the 
enumeration  of  the  leucocytes  in  a  drop  of  blood  diluted  200 
times  is  too  laborious,  and  a  further  dilution  of  the  blood 
is  advisable.  The  further  dilution  may  be  made  with  a 
Wright's  capillary  tube  (see  page  51)  in  the  following  way. 
Make  a  mark  on  the  tube  and  draw  up  to  the  mark  1  volume 
of  the  1  in  200  dilution  and  9  volumes  of  the  diluting  fluid. 
The  blood  is  now  diluted  1  in  2,000  times.  Blow  out  the 
mixture  into  a  watch  glass,  mix  thoroughly,  draw  up  5  c.mm. 
of  the  mixture  to  the  upper  mark  of  the  Strong's  pipette  and 
proceed  as  before.  The  number  of  leucocytes  counted  will 
have  to  be  multiplied  by  400  instead  of  by  40. 

(2)  The  Thoma-Zeiss  method. --The  apparatus  required 


42  CLINICAL   PATHOLOGY. 

consists  of  a  special  pipette,  a  diluting  fluid  and  a  hsemo- 
cytometer.  The  diluting  fluid  consists  of  a  0"3  per  cent, 
solution  of  acetic  acid  in  distilled  water  with  sufficient  methyl 
green  added  to  give  the  fluid  a  distinct  green  colour.  The 
acetic  water  dissolves  the  red  cells  and  the  methyl  green  stains 
the  nuclei  of  the  leucocytes.  The  blood  is  drawn  up  to  the 
0"5  mark  on  the  pipette  and  the  diluting  fluid  to  the  11  mark. 
The  pipette  is  manipulated  as  described  under  the  enumera- 
tion of  the  red  cells,  and  the  mixture  is  put  up  on  the 
haBinocytometer  slide  in  the  same  manner.  The  dilution  of 
the  blood  is  1  in  20.  All  the  leucocytes  in  the  entire  set  of  16 
large  squares  are  counted.  To  calculate  the  number  of 
leucocytes  per  c.mm.  of  undiluted  blood  multiply  the  average 
number  of  leucocytes  per  small  square  (i.e.,  the  total  number 
of  leucocytes  counted  in  the  16  large  squares  divided  by  256) 
by  20  times  4,000.  The  average  number  of  leucocytes  counted 
in  the  entire  set  of  squares  is  only  abaut  20,  and  the 
possible  error  is  considerable.  The  sole  advantage  of  this 
method  is  that  it  is  rather  more  rapid  than  Strong's 
method. 

For  the  enumeration  of  both  red  cells  and  white  cells 
Strong's  method  has  certain  advantages  over  the  Thoma-Zeiss 
technique.  The  latter  method  is  described  here  because  it  is 
sufficiently  reliable  and  is  still  widely  used. 

To  clean  pipettes. — All  blood  pipettes  should  be  cleaned 
immediately  after  use.  It  is  sufficient  to  first  suck  water  up 
and  down  the  pipettes,  then  alcohol  and  then  ether.  If 
particles  of  blood  or  dust  have  lodged  in  the  pipette  these 
should  first  be  removed  with  a  thread  of  fine  silver  wire.  If 
the  blood  has  been  allowed  to  clot  in  the  tube  immerse  the 
tube  in  33  per  cent,  acetic  acid,  removing  the  blood  with  a 
fine  wire  at  intervals  as  it  softens  ;  it  may  take  a  few  days  to 
remove  a  firm  clot,  but  the  process  may  be  considerably 
hastened  by  using  glacial  instead  of  33  per  cent,  acetic  acid. 
If  the  fine  end  of  a  pipette  is  chipped  or  notched  the  pipette  is 
broken  and  should  be  discarded. 

The  stained  blood. — The  materials  required  are  clean 
slides  or  cover- slips  and  a  blood  stain. 

If  cover-glasses  are  used  for  making  the  blood  films  it  is 
essential  that  they  should  be  clean  and  absolutely  free  from 
grease.     The  best  quality  of  square  cover-glasses  should  be 


THE   METHODS   OF   EXAMINING    THE   BLOOD.    43 

obtained  and  boiled  in  a  wide  evaporating  dish  or  a  sand  bath 
for  two  hours  in  the  following  solution  : — 

Sulphuric  acid  .         ...         .         .60  c.c. 

Potassium  bichromate        .         .         .60  grammes. 
Distilled  water  ....     1,000  c.c. 

Fresh  solution  should  be  added  from  time  to  time  as 
evaporation  occurs,  and  the  glasses  should  be  occasionally 
stirred  with  a  glass  rod.  The  glasses  should  then  be  trans- 
ferred to  distilled  water  and  washed  in  it  thoroughly  with 
several  changes.  They  should  then  be  placed  in  absolute 
alcohol,  and  when  required  for  use  picked  out  with  clean 
forceps  and  ignited  in  a  Bunsen  flame. 

Slides  should  also  be  of  good  quality,  and  can  be  cleaned  in 
the  same  manner  as  the  cover-glasses.  It  is  quite  sufficient, 
however,  to  first  rub  them  with  very  fine  emery  paper  (the 
best  emery  paper  for  the  purpose  bears  the  trade  symbol 
"  Hubert  0000  ")  and  then  to  place  them  in  absolute  alcohol. 
When  they  are  required  for  use  wipe  them  dry  with  a  clean 
cloth  and  then  warm  them  in  the  Bunsen  flame  to  drive  off 
the  last  trace  of  moisture. 

The  blood  stain  employed  should  be  either  Leishman's  or 
Jenner's  ;  both  are  excellent.  Leishman's  stain  can  be  bought 
ready  made  up,  but  the  majority  of  such  solutions  are  un- 
satisfactory. It  is  advisable  to  buy  the  stain  and  the  alcohol 
separately  and  to  make  up  in  the  following  manner : — Place 
1  gramme  of  the  stain  (Grubler)  in  a  clean,  well -stoppered 
bottle  ;  add  to  it  100  c.c.  of  the  best  methyl  alcohol.  Shake 
well  and  stand  in  a  dark  cupboard.  Shake  every  morning  for 
a  week  and  keep  for  at  least  one  month  before  using.  The 
stock  bottle  of  stain  made  in  this  manner  keeps  indefinitely  in 
ordinary  climates,  and  when  required  for  use  is  diluted  with 
an  equal  volume  of  methyl  alcohol,  the  strength  of  stain 
employed  being  then  0*5  per  cent,  in  methyl  alcohol. 

Jenner's  stain  can  be  bought  in  solution  or  made  up  as 
follows  (Strangeways) : — Two  stock  solutions  are  kept,  a  0'5 
per  cent,  solution  of  eosin  yellow  shade  (Grubler)  in  pure 
methyl  alcohol,  and  a  05  per  cent,  solution  of  medicinal 
methylene  blue  (Grubler)  in  methyl  alcohol. 

For  use  mix  eosin  solution        .         .         •     12-5  c.c. 
methylene  blue  solution         .     10'0  c.c 

In  making  up  both  this  stain  and  Leishman's  stain   it  is 


44  CLINICAL   PATHOLOGY. 

essential  that  all  bottles  and  measuring  glasses  should  be 
chemically  clean.  The  stock  bottles  should  have  well-fitting 
stoppers  and  should  be  kept  in  a  dark  place. 

To  make  the  blood  films  either  slides  or  cover-glasses 
can  be  used ;  the  latter  give  excellent  results  in  skilled 
hands  ;  slides  give  equally  good  results,  and  are  to  be  recom- 
mended to  those  who  are  not  in  constant  practice,  since  a  bad 
film  on  a  slide  is  like  the  curate's  egg ;  a  bad  film  on  a  cover- 
glass  is  useless. 

To  make  a  film  on  a  slide  place  one  end  of  the  slide  against 
the  drop  of  blood,  taking  care  not  to  touch  the  skin  of  the  ear. 
Place  the  slide  flat  on  a  smooth  firm  surface,  such  as  a  polished 


Fie.  7. — To  make  a  Blood  Film  on  a  Slide. 

table,  and  hold  it  in  position  with  the  thumb  and  first  finger 
of  the  left  hand.  "With  the  right  hand  place  the  end  of  a 
second  slide  in  the  drop  of  blood  and  hold  it  there  until  the 
blood  has  run  across  the  breadth  of  the  slides.  Draw  the 
second  slide  slowly  across  the  entire  length  of  the  first, 
maintaining  an  angle  of  about  45  degrees  between  the  two 
slides.  There  should  be  no  pressure  whatever  beiween  the 
surfaces  of  the  slides,  and  to  ensure  this  the  second  slide 
should  be  held  in  the  thumb  and  first  finger  of  the  right  hand 
at  about  the  level  of  their  distal  joints,  the  tips  of  the  fingers 
being  supported  by  the  table.  The  more  slowly  the  film  is 
made  the  thinner  the  resulting  film.  The  even  spreading  of 
the  film  is  assisted  by  previously  warming  the  slide  in  the 
flame  of  a  spirit  lamp.  As  soon  as  the  film  is  spread  the 
slide  should  be  waved  vigorously  in  the  air  to  ensure  the 
immediate  drying  of  the  film  and  thus  avoid  undue  shrinkage 
of  the  cells. 

To  make  films  on  cover-glasses  hold  two  glasses,   one  in 
each  hand,  by  their  corners.     Place  the  centre  of  one  glass 


THE    METHODS   OF   EXAMINING   THE   BLOOD.    45 

against  the  drop  of  blood.  Apply  the  centre  of  the  second 
glass  to  the  drop  on  the  first.  Hold  the  two  glasses  together 
until  the  blood  has  spread  across  the  glass.  Eapidly  separate 
the  two  glasses.  The  thinness  of  the  resulting  film  depends 
upon  the  size  of  the  drop  of  blood  and  on  the  rapidity  and 
evenness  with  which  the  glasses  are  separated.  If  the  glasses 
stick  at  all,  the  separation  has  been  unduly  delayed  and  the 
films  are  useless. 

To  stain  the  film.— (1)  With  Leishman's  stain. 

Cover  the  film  well  all  over  with  the  0*5  per  cent,  stain  and 
leave  for  30  seconds. 

Add  about  twice  the  volume  of  distilled  water  to  the  stain. 
Mix  stain  and  water  with  a  glass  pipette  until  an  iridescent 
scum  forms  over  the  surface  and  leave  for  7  minutes. 

Pour  off  the  stain  and  cover  the  film  with  distilled  water 
only  for  2  minutes. 

Wash  off  the  water  with  fresh  distilled  water,  wipe  clean 
the  back  of  the  slide,  and  gently  blot  the  film  dry  with  clean 
blotting  paper. 

To  preserve  the  film  mount  when  dry  in  Canada  balsam 
with  a  thin  cover-glass. 

The  film  is  fixed  by  the  methyl  alcohol  in  the  first  stage,  it 
is  stained  during  the  7  minutes  of  the  second  stage,  and 
the  colours  are  differentiated  by  the  distilled  water  in  the 
third  stage. 

It  is  essential  that  the  pipettes  and  beakers  used  should  be 
first  washed  out  with  distilled  water  and  that  the  water  used 
should  be  distilled.  The  water  must  be  neutral  to  litmus 
paper  and  must  give  no  precipitate  with  silver  nitrate. 

(2)  With  Jenner's  stain. 

Cover  the  film  with  the  stain  and  place  a  watch  glass  or 
inverted  dish  over  the  film  to  prevent  evaporation. 

Stain  for  4  minutes. 

Wash  in  distilled  water  until  the  film  becomes  a  delicate 
pink  tint. 

Blot  dry. 

The  differential  count  should  always  be  made  with 
a  -J^inch  objective  and  preferably  with  the  help  of  a 
mechanical  stage.  Daylight  should  be  used  when  available. 
Choose  the  thinnest  and  most  even  part  of  the  film  and  count 
as  a  minimum  number  200  leucocytes,  tabulating  on  a  piece 


46  CLINICAL   PATHOLOGY. 

of  paper  each  leucocyte  under  its  proper  heading.  In  order  to 
obtain  the  percentage  of  the  leucocytes  present  in  their  relative 
proportions  it  is  not  necessary  to  count  200  consecutive  cells 
so  long  as  care  is  taken  to  avoid  counting  any  cell  more  than 
once. 

Special  Methods  of  Examining  the  Blood. 

The  coagulation  time. — A  simple  and  accurate  method 
of  estimating  the  coagulation  time  of  the  blood  is  that  of 
Wright.  The  necessary  apparatus  consists  of  a  series  of 
capillary  tubes,  elastic  bands,  a  beaker,  a  jug  of  hot  and  a  jug 
of  cold  water,  a  watch  with  a  second  hand,  and  a  thermometer. 
The  capillary  tubes  are  of  the  same  calibre  and  are  provided 
with  a  5  c.mm.  mark.  The  procedure  is  as  follows : — Clean 
the  patient's  thumb  with  ether.  Wrap  a  piece  of  elastic  tubing 
round  the  thumb  from  its  base  to  nearly  the  tip.  Puncture 
the  tip  of  the  thumb  with  a  sterile  surgical  needle.  Draw  up 
blood  to  the  mark  on  the  pipette.  (It  is  not  essential  to 
obtain  the  exact  quantity  of  blood,  slight  variations  in  the 
amount  being  of  less  importance  than  rapidity  in  the  manipu- 
lation of  the  tubes.)  Note  the  exact  time  by  the  watch. 
Stretch  a  flat  elastic  band  over  the  ends  of  the  tube  to 
prevent  water  entering.  Stand  the  tube  in  the  beaker 
filled  with  water  at  37°  C.  Stir  the  water  occasionally  with  the 
thermometer,  and  keep  the  temperature  constant  by  adding 
hot  or  cold  water.  Prepare  3  or  4  more  capillary  tubes 
in  the  same  way,  numbering  each  tube  and  taking  the  time  of 
each.  At  the  end  of  3  minutes  take  out  the  first  tube  and 
blow  out  the  blood.  Give  the  second  tube  3 J  minutes, 
and  if  the  blood  is  still  fluid  give  the  third  tube  4 
minutes,  and  so  on.  The  tube  from  which  the  blood  fails  to 
be  expelled  by  blowing  gives  the  coagulation  time.  The 
time  is  a  very  constant  one  for  normal  people  of  about 
3^  minutes. 

The  fragility  of  the  red  cells. — The  examination  of 
the  action  of  various  strengths  of  salt  solutions  on  the  red 
cells  is  rarely  called  for.  It  is  described  here  because  it  is 
a  simple  proceeding  and  forms  a  part  of  the  examination  of 
the  blood  in  acholuric  family  jaundice.  No  special  apparatus 
is  required.  The  necessary  materials  consist  of  2  burettes, 
10  large  watch   glasses,  sodium   chloride,  distilled  water,  a 


THE    METHODS   OF   EXAMINING   THE    BLOOD.    47 

Wright's  capillary  tube  provided  with  a  rubber  teat  (page  51), 
a  number  of  small  tubes  sealed  at  one  end.  The  small  tubes 
are  readily  made  from  a  piece  of  glass  tubing  and  should  be 
about  2  inches  long. 

The  procedure  is  as  follows.  Make  up  exactly  a  1  per  cent, 
solution  of  sodium  chloride  in  distilled  water.  Fill  one 
burette  with  the  salt  solution,  the  other  with  distilled  water. 
Eun  into  the  first  watch  glass  0'9  c.c.  of  water  and  O'l  c.c. 
of  salt  solution  (=  0"1  per  cent,  salt),  into  the  second  0"8  c.c. 
of  water  and  0*2  c.c.  of  salt  (=  0'2  per  cent,  salt),  and  so 
on.  A  series  of  solutions  from  O'l  per  cent,  saline  to 
1  per  cent,  is  thus  obtained.  Deliver  an  equal  volume  of 
each  solution  into  a  double  series  of  the  small  tubes.  Stand 
the  two  series  of  tubes  in  order  in  a  Petri  dish  rilled  with 
"  plasticine."  Make  a  mark  about  2  inches  from  the  end 
of  the  Wright's  pipette.  Obtain  blood  from  the  patient's 
thumb  in  the  same  way  as  for  the  estimation  of  the  coagula- 
tion time.  Blow  out  a  volume  of  blood  into  each  tube  in  the 
series.  Bepeat  the  process  with  the  blood  of  a  normal  person 
and  the  second  series  of  tubes.  Invert  each  tube  and  stand  the 
Petri  dish  in  the  incubator  at  37°  C.  for  30  minutes.  If  no 
incubator  is  available  the  tubes  may  be  stood  in  warm  water 
or  even  left  at  room  temperature.  In  those  tubes  in  which 
haemolysis  occurs  the  supernatant  fluid  is  tinged  red  and  there 
is  no  deposit  of  red  cells.  Where  haemolysis  is  absent  the 
salt  solution  remains  colourless  and  the  red  cells  form  a 
deposit  at  the  bottom  of  the  tube. 

Other  methods. — Other  special  methods  of  examination 
of  the  blood,  such  as  the  estimation  of  the  alkalinity  of 
the  blood,  and  the  presence  of  fat  or  bile  in  the  serum, 
are  described  under  the  heading  of  the  chemistry  of  the 
blood. 

Certain  special  methods  commonly  described  and  extremely 
rarely  practised  are  omitted  altogether.  Such  methods 
include  estimations  of  the  viscosity  of  the  blood,  and  are  of 
some  scientific  but  of  no  clinical  value. 


CHAPTEK  IV. 

THE  BLOOD  SEBUM — AGGLUTININS  AND  OPSONINS. 

The  nature  of  agglutinins. — Agglutinins  are  antibodies 
appearing  in  the  blood  in  excess  in  response  to  infection.  The 
more  important  agglutinins  are  those  which  act  upon  bacteria, 
and  these  have  the  property  of  checking  the  motility  of 
organisms  and  causing  them  to  come  together  into  clumps  or 
masses.  The  phenomenon  is  a  very  striking  one  to  watch, 
but  its  purpose  and  mode  of  action  are  obscure.  The  bacterial 
clumps  produced  by  an  agglutinating  serum  acting  on  a 
suspension  of  organisms  are  not  permanent,  and  the  organisms 
themselves  are  only  temporarily  embarrassed,  for  after  an 
interval  the  clumps  break  up  again  into  separate  individuals, 
the  bacteria  regain  their  motility  and  are  capable  of  multi- 
plying and  producing  disease.  Agglutinating  substances  may 
also  be  present  in  the  serum  which  have  no  action  upon 
bacteria,  but  are  capable  of  agglutinating  red  blood  corpuscles ; 
such  bodies  are  known  as  hemagglutinins. 

The  agglutinins  are  thermo-stable,  that  is  to  say,  an  aggluti- 
nating serum  heated  to  60°  C.  is  still  capable  of  clumping 
bacteria  or  red  cells. 

In  common  with  other  antibodies,  the  agglutinins  are 
specific,  and  act  only  upon  the  infecting  agent  or  "  antigen  " 
which  calls  them  into  existence.  The  agglutinin  for  the  typhoid 
bacillus,  for  example,  has  the  power  of  clumping  that  bacillus 
and  no  other  bacteria.  It  happens,  however,  that  infection  by 
one  organism  not  infrequently  leads  to  a  general  increase  in 
the  antibodies  for  other,  and  particularly  for  closely  allied 
organisms,  the  increase  in  the  specific  antibody  being  only 
relatively  greater  than  the  increase  in  the  general  antibodies. 
In  any  febrile  condition,  such  as  tuberculosis  or  influenza,  there 
may  be  a  considerable  increase  in  the  amount  of  agglutinin  for 
the  typhoid  bacillus.  In  paratyphoid  infections  there  is  almost 
always  a  considerable  increase  in  the  agglutinin  for  the  typhoid 
bacillus,  but  in  typhoid  infections  the  increase  in  the  typhoid 


BLOOD   SEEUM— AGGLUTININS   AND   OPSONINS    49 

agglutinin  is  appreciably  greater  than  that  found  in  any  other 
circumstances.  Further,  agglutinins  are  present  in  the  blood 
in  health,  but  in  minute  quantities ;  they  are  enormously 
increased  in  response  to  infections.  In  making  use  of 
agglutinins  in  the  clinical  diagnosis  of  disease  it  is  evidently 
insufficient  to  demonstrate  their  presence ;  it  is  essential  to 
estimate  their  amount.  The  amount  of  agglutinin  in  any 
serum  is  judged  by  progressively  diluting  the  serum  until  the 
greatest  dilution  capable  of  agglutinating  the  bacteria  is  arrived 
at. 

The  use  of  agglutinins  in  diagnosis. — Some  infecting 
agents,  such  as  the  tubercle  bacillus,  do  not  lead  to  any  appre- 
ciable production  of  agglutinin  in  the  blood,  and  the  agglutina- 
tion test  is  therefore  inapplicable  as  a  mode  of  diagnosis.  In 
other  diseases,  such  as  cholera,  the  specific  agglutinin  is  manifest 
only  after  recovery  from  the  attack.  Other  organisms,  such  as 
the  bacillus  pyocyaneus,  agglutinate  spontaneously  in  saline 
suspensions.  Other  organisms  again  have  either  not  been  as 
yet  discovered  or  are  unable  to  be  cultivated  outside  the  body. 
The  agglutination  test  has  therefore  a  comparatively  limited 
field  of  application,  and  is  mainly  confined  to  the  diagnosis  of 
typhoid  and  paratyphoid  infections,  dysentery,  and  Malta 
fever. 

The  agglutinins  in  typhoid  fever. — The  estimation  of  the 
typhoid  agglutinin  is  known  as  the  Griinbaum- Widal  reaction 
and  is  of  the  greatest  value  in  diagnosis.  A  positive  reaction  is 
one  of  the  very  few  pathognomonic  physical  signs  in  medicine, 
since  it  indicates  with  certainty  the  presence  of  an  infection 
with  the  typhoid  bacillus.  If  we  except  the  comparatively 
rare  examples  of  "  typhoid  carriers,"  a  positive  reaction  is 
almost  certain  evidence  of  typhoid  fever.  A  classical  case  of 
typhoid  fever  can  be  safely  diagnosed  on  clinical  grounds 
alone,  but  the  disease  is  so  commonly  atypical  that  the  diag- 
nosis is  occasionally  made  and  is  frequently  confirmed  by 
means  of  the  agglutination  test.  A  negative  reaction  does  not 
contraindicate  typhoid  fever,  since  it  commonly  happens  that 
the  increase  in  agglutinin  is  insufficient  for  a  completely 
positive  reaction,  and  a  partial  reaction  is  not  conclusive 
evidence  of  typhoid  fever.  A  completely  negative  reaction, 
however,  is  strongly  opposed  to  the  diagnosis  of  typhoid  fever. 
A  positive  reaction  is  rarely  obtained  before  the  end  of  the 

p.  4 


50  CLINICAL   PATHOLOGY. 

first  or  the  beginning  of  the  second  week  of  the  disease,  but 
an  appreciable  increase  in  the  agglutinins  is  usual  within  the 
first  4  or  5  days  of  the  onset  of  symptoms.  The  reaction 
usually  remains  positive  throughout  the  course  of  the  disease, 
and  for  some  weeks,  or  even  a  few  months,  after  the  tempera- 
ture has  become  normal.  A  partial  reaction  may  persist  for  a 
year  or  more. 

The  reaction  is  of  no  particular  value  as  a  basis  for  prog- 
nosis ;  a  mild  case  of  typhoid  fever  may  never  give  a  strongly 
positive  agglutination  test,  and  fatal  cases  may  or  may  not 
react  strongly. 

In  the  very  early  stages  of  typhoid  fever  the  diagnosis  may 
be  made  by  the  isolation  of  the  bacilli  from  the  blood  (page  83) ; 
in  the  later  stages,  if  the  agglutination  test  is  doubtful,  the 
bacilli  rnsij  be  obtained  from  the  faeces  (page  342),  or  less 
frequently  from  the  urine  (page  282). 

The  technique  of  the  Griinbaum-Widal  test. —The 
materials  required  for  the  performance  of  the  reaction  are  as 
follows : — Serum  tubes,  Wright's  capillary  tubes  and  rubber 
teat,  hollow-ground  slides  and  vaseline,  cover-glasses,  normal 
saline,  watch  glasses,  a  bowl  or  small  hand-basin  containing 
carbolic  acid  (1  in  20),  and  a  24  hours  old  culture  of  typhoid 
bacilli  on  an  agar  slope. 

The  "Wright's  tubes  and  the  serum  tubes  can  readily  be 
made  from  glass  tubing  with  the  aid  of  a  blow-pipe  ;  the  glass 
tubing  should  have  an  outside  diameter  of  \  inch. 

To  make  a  Wright's  tube  hold  the  glass  tubing  with  one 
hand  at  each  end  and  heat  it  to  a  red  heat  as  near  to  one 
end  as  it  is  convenient  to  hold  it,  continually  rotating  the 
glass.  As  soon  as  the  heated  portion  is  freely  malleable 
remove  it  from  the  flame  and  separate  the  two  ends  evenly, 
without  force  and  moderately  slowly.  Fuse  the  terminal 
portion  of  the  capillary  part  of  the  tube  in  the  flame.  As 
soon  as  the  glass  is  cool  enough  to  hold  repeat  the  process 
and  make  a  series  of  tubes.  Leave  both  ends  of  the  tube 
sealed  until  required  for  use,  then  nick  each  end  with  a  file 
and  break  them  off.  The  bulbous  portion  of  each  tube  should 
be  about  \\  inches  long  and  the  capillary  portion  about  9 
inches.  The  bore  of  the  capillary  part  should  be  nearly  equal 
throughout,  tapering  very  slightly  towards  the  distal  end. 
The  tubes  are  readily  made  with  a  little  practice,  the  points 


BLOOD    SERUM— AGGLUTININS   AND   OPSONINS.  51 

requiring  experience  being  the  size  of  the  blow-pipe  flame,  the 
degree  to  which  the  glass  is  heated,  and  the  rapidity  with 
which  the  capillary  tube  is  drawn  out.  The  tendency  is  either 
to  heat  the  glass  insufficiently  and  produce  a  short,  thick  tube, 
or  to  draw  the  ends  apart  too  rapidly  and  make  the  tube 
excessively  long  and  thin. 

The  serum  tubes  for  collecting  the  blood  are  made  in  a 
similar  manner,  but  the  bulbous  portion  is  left  longer  (about 
2  inches),  and  the  capillary  part  is  made  shorter  and  burnt 
through  at  its  centre,  the  distal  half  being  left  to  provide  the 
capillary  portion  of  the  next  tube  drawn  out. 

To  obtain  the  serum  cleanse  the  patient's  thumb  with 
ether  and  let  it  dry.  If  the  hand  is  cold  place  it  previously  in 
hot  water  and  dry  thoroughly.  Wind  a  piece  of  fine  rubber 
tubing  round  the  thumb  from  the  base  nearly  to  the  nail. 
With  a  surgical  needle  make  a  sharp,  deep  stab  at  the  side  of 


Pig.  8.— Serum  Tube ;  Wright's  Tube  ;  Eubber  Teat. 

the  thumb  in  the  line  of  the  digital  artery.  Avoid  the  pulp  of 
the  thumb.  Having  broken  off  both  ends  of  the  serum  tube, 
hold  one  end  lightly  in  the  drop  of  blood,  sloping  the  tube  a 
very  little  downwards.  Keep  rotating  the  tube.  When  the 
blood  has  all  entered  the  tube  lay  the  tube  gently  down  on 
a  flat  surface,  loose  the  rubber  tubing,  rub  the  thumb  briskly 
with  a  dry  swab,  reapply  the  tubing,  and  continue  to  fill  the 
tube.  When  the  bulbous  portion  of  the  tube  is  about  half 
full,  wipe  the  end  containing  the  blood  and  seal  it  off  in  a 
flame,  then  seal  off  the  other  end.  Stand  the  tube  in  an 
upright  position  for  about  half  an  hour  or  until  the  serum  has 
separated,  then  centrifuge  the  tube  at  a  moderate  speed.  (For 
the  purposes  of  this  reaction  a  comparatively  small  quantity 
of  serum  is  required,  and  the  centrifuging  may  frequently  be 
dispensed  with.) 

The  slides  for  the  hanging  drops  should  be  provided 
with  a  central  circular  depression,  but  this  is  not    essential. 

4—2 


52  CLINICAL    PATHOLOGY. 

To  prepare  the  slides,  warm  them  in  the  Bunsen  flame,  take 
up  a  little  vaseline  on  a  glass  rod,  warm  the  rod  in  the  flame 
to  melt  the  vaseline,  and  then  draw  the  end  of  the  rod  round 
the  circular  depression  in  the  slide,  leaving  a  substantial  ring 
of  vaseline.  No  vaseline  should  be  allowed  to  drop  into  the 
central  depression. 

The  typhoid  culture  used  is  of  considerable  importance.  A 
reliable  strain  can  be  obtained  from  any  known  laboratory,  or  the 
bacilli  may  be  isolated  from  the  blood  of  a  case  of  early  typhoid 
fever  (page  83),  or  from  the  spleen  post  mortem  (page  161). 
From  whatever  source  the  bacilli  are  derived  the  culture 
should  be  examined  in  two  ways — the  full  cultural  characters 
of  the  bacillus  should  be  investigated  (page  135)  and  the  bacillus 
should  be  tested  against  a  known  positive  serum.  The 
readiness  with  which  various  strains  of  bacilli  are  aggluti- 
nated is  not  constant,  and  a  bacillus  is  exceptionally  met  with 
which  has  all  the  cultural  characters  of  the  typhoid  bacillus, 
but  which  is  not  agglutinated  by  the  most  powerful  sera.  A 
reliable  strain  of  bacilli  once  obtained  can  often  be  sub- 
cuitured  over  an  almost  indefinite  period  without  change  of 
character.  The  subculture  used  for  performing  the  test 
should  have  been  incubated  from  the  previous  day  on  an  agar 
slope.  Before  making  the  suspension  put  up  a  subculture 
from  the  agar  slope  into  broth  to  preserve  the  strain.  To 
make  the  suspension  add  warm  saline  to  nearly  halfway 
up  the  agar  slope  and  replace  the  cotton-wool  plug.  Gently 
shake  the  tube  until  the  growth  is  washed  off  into  the  saline 
and  the  latter  has  become  distinctly  milky.  It  may  be  neces- 
sary to  assist  the  washing  off  of  the  growth  into  the  saline  by 
gently  rubbing  the  former  with  a  sterile  platinum  wire.  When 
a  milky  suspension  has  been  obtained,  remove  the  cotton-wool 
plug  and  drop  it  in  the  carbolic  bowl.  Filter  the  suspension 
through  a  small  filter  paper  moistened  with  warm  saline  and 
held  in  a  pair  of  forceps  over  a  watch  glass.  Place  the  culture 
tube  and  filter  paper  in  the  carbolic  and  sterilise  the  ends  of 
the  forceps  in  the  Bunsen  flame.  The  object  of  filtering  the 
suspension  is  to  remove  any  clumps  of  bacilli  which  may  be 
washed  off  as  such  from  the  surface  of  the  medium.  The 
discrete  bacilli  pass  through  the  filter  paper. 

The  reaction  is  then  performed  as  follows  : — With  a  blue 
glass-pencil  (or  with  a  pen  and  ink)  make  a  mark  on  a  Wright's 


BLOOD    SERUM— AGGLUTININS   AND   OPSONINS.  53 

pipette  about  half  an  inch  from  the  end.     By  means  of  the 
rubber  teat,  which  must  fit  tightly  to  the  pipette,  draw  up  a 
volume  of  the  blood  serum  to  be  tested  to  the  mark,  admit  a 
column  of  air,  and  draw  up  9  equal  volumes  of  normal  (0*85 
per  cent.)  salt  solution,  leaving  a  small  column  of  air  between 
each  volume.     Blow  out  serum  and  saline  into  a  clean  watch 
glass  and  mix  thoroughly  ;  the  serum  is  now  diluted  to  1  in  10. 
Draw  up  a  volume  of  the  diluted  serum  to  the  mark  and  an 
equal  volume  of  the  suspension  of  bacilli.     Mix  in  a  watch 
glass  and  transfer  a  sample  drop  with  the  pipette  to  the  centre 
of  a  cover-glass,  spreading  out  the  drop  so  as  to  cover  about 
one-fourth  the  area  of  the  glass.     Pick  up  the  cover-glass  by 
pressing  on  it  one  of  the  vaseline  ringed  slides.      Turn  the 
slide    over.      Press   down   the   cover-glass  with    a   mounted 
needle  over  the  whole  circumference  of  the  vaseline  ring  so 
that  no  air  can  gain  admittance.     No  vaseline  should  touch 
the  hanging  drop.     Mark  on  the  slide  the  time  and  the  dilution 
of  the  serum  (1  in  20).     Take  another  volume  of  the  diluted 
serum  in  saline  and  4  volumes  of  the  suspension  of  bacilli. 
Mix   and  prepare  a  hanging   drop  as  before.      The  dilution 
of  the  serum  is  now  1  in  50.     Take  a  third  volume  of  the 
diluted  serum  and  9  volumes  of  the  bacillary  suspension  and 
prepare  a  hanging  drop  in  which  the  serum  is  diluted  1  in 
100.      Make  a  fourth  drop  from    a  sample    of  the   typhoid 
suspension  to  which  no  serum  has  been  added  and  label  the  slide 
"  control."     Examine  each  slide  with  the  microscope  vertical 
and  a  -jt  inch  objective,  altering  the  aperture  of  the  diaphragm 
until  the  refractile  bacilli  are  clearly  seen.    Observe  at  intervals 
the  motility  of  the  bacilli  and  the  formation  of  clumps.     Com- 
pare the  drops  containing  serum  with  the  control ;  if  the  bacilli 
in  the  latter  lose  their  motility  or  come  together  in  clumps 
the  test  is  valueless ;  it  is,  however,  extremely  rare  to  meet 
with  a  sample  of  typhoid  bacilli  which  agglutinate  spontaneously 
in  suspension.      In  a  positive  reaction  between  serum  and 
bacilli  the  latter  become  motionless  and  collected  into  com- 
pact masses  easily  visible  to  the  naked  eye,  few  if  any  bacilli 
remaining  isolated  between  the  clumps.    After  completing  the 
reaction  place  the  slides  (after  partially  slipping  off  the  cover- 
glasses),  the  capillary  tubes,  typhoid  suspension,  watch  glasses, 
and  everything  which  can  possibly  have  come  in  contact  with 
the  bacilli  into  the  carbolic  basin  and  leave  them  there  till 


54  CLINICAL   PATHOLOGY. 

the  next  day.  Make  sure  that  no  bacilli  have  been  spilt  on 
the  bench,  but  if  they  have  soak  the  bench  in  carbolic.  Wash 
your  hands  after  completing  the  reaction  and  do  not  smoke 
while  it  is  being  performed.* 

The  interpretation  of  the  reaction  is  based  upon  the 
time  taken  for  the  completion  of  the  reaction  and  upon  the 
dilution  of  serum  capable  of  producing  agglutination  of  the 
bacilli.  "With  serum  very  loaded  "with  agglutinins  clumping 
ma}^  take  place  within  a  few  minutes  in  the  1  in  100  dilution. 
In  a  completely  negative  case  no  clumping  occurs  in  the  1  in 
20  dilution,  and  the  motility  of  the  bacilli  may  even  be 
accelerated.  Complete  clumping  in  the  1  in  20,  partial 
clumping  with  incomplete  loss  of  motility  in  the  1  in  50,  and 
little  or  no  reaction  in  the  1  in  100  is  a  "  partial  "  reaction. 
The  essentials  of  a  positive  reaction  are  that  clumping  and  loss 
of  motility  should  be  complete  in  the  1  in  50  dilution  within  half 
an  hour.  Such  a  reaction  is  the  strongest  possible  evidence  of 
typhoid  fever.  Complete  absence  of  reaction  in  the  1  in  20 
dilution  is  strongly  opposed  to  the  diagnosis  of  typhoid  in  a 
febrile  case  of  any  duration.  A  partial  reaction  is  often  of 
assistance  when  taken  in  conjunction  with  the  physical  con- 
dition of  the  patient,  and  in  cases  of  doubt  should  be  repeated 
after  a  few  days'  interval. 

It  may  be  stated  here,  and  cannot  be  too  strongly  emphasised, 
that  the  deductions  drawn  from  any  pathological  test,  even 
from  the  Wassermann  or  Griinbaum-Widal  reactions,  should 
never  be  made  from  the  test  alone.  The  interpretation  of  the 
results  requires  a  knowledge  of  the  clinical  condition  of  the 
patient  coupled  with  an  understanding  of  how  the  test  is 
performed  and  what  it  means. 

Other  methods  of  performing  the  reaction  are  in  fairly 
common  use ;  the  method  described  above  is  perhaps  the  one 
most  widely  employed,  and  is  known  as  the  microscopical 
method.  Other  observers  prefer  the  macroscopic  test,  which 
is  performed  by  mixing  diluted  serum  and  a  saline  suspension 
of  bacilli  in  a  Wright's  tube ;  sealing  the  end  in  a  flame,  and 

*  It  may  be  said  generally  of  smoking  in  a  laboratory  that  the  atmo- 
sphere of  such  places  is  sufficiently  vitiated  without  indulging  in  this 
habit.  If  smoking  is  permitted  care  should  be  taken  never  to  lay  down 
a  pipe  or  cigarette  on  the  bench,  since  there  is  a  distinct  risk  of  transferring 
organisms  from  the  bench  to  the  mouth. 


BLOOD    SERUM— AGGLUTININS   AND   OPSONINS.  55 

standing  in  a  vertical  position  either  at  room  temperature  or 
in  an  incubator  at  37°  C.  In  a  positive  reaction  a  granular 
precipitate  of  clumped  bacilli  forms  in  the  capillary  tube  and 
sinks  towards  the  bottom  of  the  tube;  the  control  tube  con- 
taining bacilli  in  salt  solution  remains  uniformly  turbid.  The 
interpretation  of  the  results  depends  upon  the  dilution  of  the 
serum,  the  time  during  which  the  precipitate  forms,  and  the 
temperature  at  which  the  reaction  takes  place.  The  method 
is  a  perfectly  reliable  one. 

The  living  bacilli  may  be  substituted  by  a  suspension  of 
dead  organisms  such  as  can  be  obtained  ready  for  use  from 
various  sources.  The  dead  bacilli  ready  prepared  are  more 
convenient  for  those  not  constantly  working  in  a  laboratory, 
but  should  only  be  used  when  unavoidable  and  never  without 
adequate  controls,  since  such  preparations  have  been  provided 
in  some  instances  with  the  bacilli  omitted  and  others  with  the 
organisms  already  clumped. 

Certain  ingenious  little  automatic  test  cases  are  provided  by 
manufacturers  for  the  performance  of  this  reaction.  I  have  only 
examined  one  apparatus  of  this  nature  and  found  it  worthless. 
All  such  mechanical  contrivances  are  apt  to  deceive  and  should 
be  avoided. 

The  agglutinins  in  diseases  other  than  typhoid 
fever. — Paratyphoid  infections  are  infections  not  by  the 
typhoid  bacillus,  but  by  organisms  closely  allied  to  it  and 
known  as  paratyphoid  bacilli.  These  bacilli  are  of  more  than 
one  variety,  the  organism  most  commonly  met  with  in  this 
country  being  that  known  as  b.  paratyphosus  B.  The  para- 
typhoid bacilli  give  rise  to  a  clinical  condition  indistinguishable 
from  typhoid  fever  by  other  than  bacteriological  methods. 
The  paratyphoid  infections,  however,  tend  to  run  a  milder 
course. 

Infection  by  one  of  the  paratyphoid  bacilli  may  be  sus- 
pected when  a  case  clinically  resembling  typhoid  fever  fails  to 
give  a  positive  serum  test.  In  such  cases  the  serum  should 
be  tested  against  stock  cultures  of  one  or  more  of  the  para- 
typhoid bacilli,  and  in  addition  attempts  should  be  made  to 
isolate  the  causative  organism  from  the  blood,  the  faeces  or 
the  urine.  The  methods  of  testing  the  agglutinating  content 
of  the  serum  or  of  isolating  the  bacillus  are  identical  with  those 
employed  in  the    case  of    the   typhoid   bacillus.     It  usually 


56  CLINICAL   PATHOLOGY. 

happens,  however,  that  the  serum  of  a  paratyphoid  infection 
agglutinates  to  a  considerable  extent  typhoid  bacilli,  and  the 
serum  must  be  diluted  beyond  the  point  at  which  one  organism 
is  agglutinated  but  not  the  other.  The  separation  of  the 
agglutinin  for  each  bacillus  is  somewhat  beyond  the  scope  of 
ordinary  clinical  methods.  It  may  be  done  as  follows  : — A  thick 
suspension  of  typhoid  bacilli  is  added  to  a  portion  of  the  serum 
and  the  mixture  incubated.  After  incubation  the  mixture  is 
centrifuged  at  a  high  speed  and  the  clear  serum  pipetted  off 
from  the  bacilli.  The  agglutinating  property  of  the  serum  is 
then  tested  in  the  usual  manner  with  paratyphoid  bacilli.  The 
agglutinins  for  the  typhoid  bacilli  have  been  removed  by  the 
previous  saturation  of  the  serum  with  these  bacilli,  and  the 
agglutinins  for  the  paratyphoid  bacilli  are  unaffected.  A  second 
sample  of  serum  is  saturated  with  paratyphoid  bacilli  and  then 
tested  on  typhoid  bacilli. 

Dysentery  of  the  bacillary  variety  and  such  of  the 
intestinal  affections  as  may  be  caused  by  one  or  other  of  the 
dysentery  bacilli  give  rise  to  specific  agglutinins  in  the  blood. 
The  agglutination  test  with  the  serum  of  dysenteric  patients  is 
conducted  in  exactly  the  same  manner  as  is  the  Griinbaum- 
Widal  reaction.  The  amount  and  the  activity  of  the  agglutinin 
present  in  dysentery  commonly  fall  below  that  produced  in 
typhoid  fever,  and  it  is  exceptional  to  meet  with  a  serum  capable 
in  dilution  of  1  in  50  of  completely  agglutinating  one  of  the 
strains  of  dysentery  bacilli  in  less  than  one  hour.  The 
dysentery  bacilli  differ  from  each  other  in  a  manner  similar  to 
the  paratyphoid  bacilli ;  the  most  widespread  infecting  agents 
of  bacillary  dysentery  are  those  first  described  by  Shiga  in 
Japan  and  Flexner  in  America.  Cases  of  amoebic  dysentery 
give  no  reaction  in  their  serum  with  the  dysentery  bacilli. 

Malta  fever  is  a  disease  produced  by  a  coccus,  the  micro- 
coccus melitensis,  and  the  serum  of  infected  persons  or  animals 
acquires  the  property  of  agglutinating  this  organism.  The 
serum  test  is  a  very  valuable  one  in  assisting  diagnosis,  and  is 
performed  in  the  same  manner  as  the  typhoid  test.  The  Malta 
fever  coccus  can  be  obtained  from  the  urine  of  an  infected 
person  or  animal,  or  from  the  blood.  If  no  case  is  available  a 
culture  can  be  obtained  from  a  reliable  laboratory,  but  it  is 
advisable  in  performing  this  reaction  that  the  culture  used 
should  be  one  comparatively  recently  isolated  from  the  body. 


BLOOD   SEBUM— AGGLUTININS   AND   OPSONINS    57 

The    agglutinins    as    evidence    of    infection.— The 

isolation  of  an  organism  from  some  part  of  the  body  is  not  of 
itself  evidence  that  the  organism  is  actually  producing  disease. 
It  may  also  happen  that  a  bacterium,  not  generally  recognised  as 
capable  of  producing  disease,  or  a  variety  of  bacteria  amongst 
which  the  infective  organism  is  in  doubt,  may  be  isolated  in 
cultures  from  a  lesion,  or  from  the  excreta,  or  from  one  of  the 
body  fluids.  In  such  cases,  and  particularly  when  the 
organism  in  question  is  a  member  of  the  colon  or  typhoid 
group,  evidence  of  the  infectivity  of  the  bacterium  may  be 
sought  for  by  examining  the  agglutinating  power  of  the 
patient's  serum  upon  it,  together  with  that  of  the  serum  from 
a  normal  person.  The  presence  of  agglutinins  in  the  serum 
in  greater  than  the  normal  amount  for  the  organism  isolated 
is  definite  evidence  of  actual  infection  by  that  organism. 
Absence  of  agglutinin  for  the  organism  is  no  evidence  against 
its  infectivity,  since  some  bacteria  give  rise  to  little  or  no 
agglutinin  in  the  blood  even  when  they  are  undoubtedly 
producing  disease.  Infection  of  the  urinary  tract  with  the 
bacillus  coli,  for  example,  commonly  produces  no  appreciable 
rise  in  agglutinin  for  this  bacillus. 

The  agglutinins  as  a  test  for  an  organism. — Just  as 
a  patient  with  an  unknown  disease  may  be  proved  to  be 
infected  with  a  known  organism  by  the  demonstration  of 
agglutinins  in  his  serum  for  that  organism,  so  a  reversal  of 
the  process,  the  testing  of  an  unknown  organism  with  a  known 
serum,  may  be  adduced  as  a  proof  of  the  nature  of  the  organism. 
A  bacillus,  isolated  from  the  blood  in  the  first  few  days  of  a 
febrile  attack,  may  be  immediately  tested  with  the  serum  of  a 
patient  known  to  have  typhoid  fever,  or  with  the  serum  of  an 
animal  which  has  been  immunised  to  the  typhoid  bacillus. 
A  positive  diagnosis  can  thus  be  made  at  an  earlier  date  than 
would  be  possible  if  the  recognition  of  the  bacillus  depended 
upon  its  full  cultural  characters.  The  reaction  is  most  com- 
monly used  in  typhoid  fever,  and  it  is  convenient  to  have  at 
hand  a  strongly  agglutinating  serum  of  this  nature.  When 
such  a  sample  of  blood  is  obtained  it  should  be  centrifuged  and 
the  clear  serum  pipetted  off"  into  a  sterile  capsule.  The  capsule 
should  be  sealed,  heated  in  water  at  56°  C.  for  half  an  hour,  and 
then  kept  in  a  cupboard,  or  preferably  on  ice.  A  serum  will  keep 
its  agglutinating  property  for  several  weeks  or  even  months. 


58  CLINICAL   PATHOLOGY. 

Hemagglutinins. — The  agglutinins  described  above  are 
those  which  act  upon  bacteria.  There  may  also  be  present 
in  the  serum  bodies  capable  of  agglutinating  red  cells,  or 
heernagglutinins.  The  hemagglutinins  have  been  already 
referred  to  under  the  description  of  the  blood  in  purpura. 
The  phenomenon  most  commonly  met  with  is  that  the  serum 
of  the  affected  person  has  the  power  of  agglutinating  washed 
normal  red  cells,  but  not  his  own  red  cells,  while  his  own  red 
cells  are  not  acted  upon  by  a  similar  agglutinating  serum. 
The  reaction  is  performed  by  mixing  in  a  Wright's  tube  1 
volume  of  the  patient's  serum  and  1  volume  of  a  10  per  cent, 
suspension  of  normal  washed  red  cells  in  normal  saline 
(page  60),  incubating  for  15  minutes  at  37°  C,  blowing  out 
the  mixture  on  to  a  slide,  and  placing  a  cover-slip  on  the 
expelled  drop.  The  red  cells  will  be  seen  to  have  run  together 
into  large,  tight  clumps.  Very  rarely  a  serum  will  be  found  to 
have  the  property  of  agglutinating  its  own  red  cells,  a 
phenomenon  which  can  hardly  take  place  in  the  body,  yet  it 
may  be  seen  to  have  occurred  immediately  the  blood  is  shed. 
On  making  a  puncture  into  the  ear  in  such  a  case  clear  serum 
exuded,  followed  by  a  clump  of  red  cells,  and  it  was  found 
almost  impossible  to  make  films  and  quite  impossible  to  make 
a  count  of  the  red  cells. 

Opsonins. 

The  nature  of  opsonins. — Opsonins  were  shown  by 
Wright  to  be  substances  present  in  the  blood  which  have  the 
property  of  acting  upon  bacteria  in  such  a  manner  that  the 
phagocytes  are  able  to  ingest  them.  Opsonins  are  present  in 
considerable  amount  in  health  ;  they  may  be  either  increased 
or  diminished  as  the  result  of  infection.  If  the  body  reacts 
favourably  to  infection,  the  opsonins  are  increased :  if  the 
infection  gains  the  upper  hand,  the  opsonins  are  diminished. 
The  fluctuations  in  the  opsonic  content  of  the  blood  in  disease 
are  due  to  alterations  partly  in  the  specific  opsonin,  that  is  the 
opsonin  which  acts  only  on  the  specific  infecting  agent,  and 
partly  in  the  general  opsonins  capable  of  acting  on  organisms 
other  than  the  infecting  agent.  Opsonins  are  destroyed  by 
heating  the  serum  to  60°  C.  and  also  by  keeping  it  ;  they  thus 
differ  from  the  agglutinins,  and  closely  resemble  the  complex 
ment  of  normal  serum, 


BLOOD   SERUM— AGGLUTININS   AND   OPSONINS.  59 

The  estimation  of  the  opsonic  content  of  the  serum  has 
been  made  use  of  as  a  method  of  diagnosis  and  as  a  means  of 
controlling  the  treatment  of  disease  by  the  administration  of 
vaccines.  The  ratio  of  the  opsonic  content  of  a  patient's 
serum  to  that  of  a  normal  serum  is  known  as  the  opsonic 
index. 

The  technique  of  the  opsonic  index  test. — The 
materials  required  are  Wright's  capillary  pipettes,  serum 
tubes,  a  suspension  of  the  organism  to  be  tested  in  normal 
saline,  the  patient's  serum,  normal  serum  and  washed  normal 
leucocytes,  normal  salt  solution  and  normal  citrated  salt 
solution,  an  incubator,  a  centrifugal  machine,  watch  glasses, 
slides,  staining  reagents,  etc. 

The  capillary  pipettes  and  serum  tubes  are  of  the  same 
nature  as  those  used  for  the  Grunbaum-Widal  test. 

The  bacterial  suspension  is  made,  when  practicable, 
from  a  '24  hours  old  culture  on  agar  of  the  organism  to  be 
tested.  The  growth  is  washed  off  the  agar  slope  by  means  of 
normal  salt  solution  and  transferred  to  a  clean  centrifuge  tube. 
The  tube  is  centrifuged  at  a  moderate  speed,  and  the  super- 
natant fluid  (free  from  clumps  of  bacteria)  is  poured  off  and 
diluted  with  saline  until  a  suspension  of  suitable  turbidity  is 
obtained.  The  strength  of  the  suspension  is  a  matter  of  much 
importance  and  can  be  gauged  by  the  degree  of  turbidity,  but 
only  as  the  result  of  experience.  Suspensions  of  organisms 
readily  ingested  by  the  phagocytes  (such  as  the  staphylococcus 
aureus)  should  be  made  thinner  than  suspensions  of  organisms 
less  readily  ingested  (such  as  the  bacillus  coll).  The  natural 
tendency  is  to  make  the  suspensions  stronger  than  is  desirable. 
In  the  case  of  the  tubercle  bacillus  it  is  convenient  to  use  the 
dried  bacilli  sterilised  by  heat.  A  small  portion  of  the  growth 
is  rubbed  up  in  a  mortar  with  saline  and  then  centrifuged  and 
treated  in  the  manner  described  above.  The  majority  of 
organisms  are  best  used  in  the  living  state,  since  heat 
sufficient  to  destroy  them  may  lead  to  a  diminution  in  their 
affinity  for  the  ordinary  stains.  All  suspensions  should  be 
freshly  made  for  each  series  of  observations,  and  should  be 
thoroughly  mixed  before  use. 

The  serum  is  obtained  in  the  same  way  as  for  the  agglutinin 
test.  In  the  case  of  the  normal  serum  it  is  advisable  to  take 
the  blood  from  several  normal  persons  and  mix  the  sera  so 


60  CLINICAL   PATHOLOGY. 

obtained  in  one  capsule  in  order  to  diminish  the  risk  of  taking 
as  normal  the  serum  of  an  infected  person.  In  the  case  of 
the  tuberculous  opsonic  index  such  a  normal  "pooled"  serum 
is  almost  essential.  The  sera  used  should  have  been  obtained 
not  more  than  24  hours  previously. 

The  washed  normal  leucocytes  are  obtained  by  pricking 
the  thumb  and  allowing  the  blood  to  drop  into  a  centrifuge 
tube  partly  rilled  with  citrated  salt  solution  having  the 
composition  of  *85  grammes  sodium  citrate  and  "85  grammes 
sodium  chloride  in  100  c.c.  of  distilled  water.  After  every 
few  drops  of  blood  have  been  added  the  tube  should  be 
inverted  in  order  to  mix  adequately  the  blood  and  the  solution. 
The  tube  is  then  centrifuged  and  the  supernatant  fluid  pipetted 
off.  Normal  salt  solution  is  added  to  the  deposit  of  red  cells 
and  leucocytes  and  the  tube  inverted  two  or  three  times.  The 
tube  is  again  centrifuged,  the  saline  removed  and  fresh  added, 
and  the  process  again  repeated.  A  final  deposit  of  washed 
blood  cells,  the  upper  layers  of  which  are  particularly  rich  in 
leucoc}7tes,  is  thus  obtained. 

The  centrifugal  machine  employed  must  be  one  capable 
of  starting  slowly,  running  smoothly,  and  stopping  gradually. 
With  a  badly-running  machine  the  leucocytes  maybe  broken  up 
and  rendered  useless  for  phagocytic  work.  The  usual  varieties 
of  hand-driven  machines  are  for  this  reason  unsuitable,  and 
some  more  expensive  apparatus  is  desirable.  The  best  form 
of  centrifuge  is  one  in  which  the  carriers  are  held  in  a  circular 
disc  or  plate,  which  can  run  free  when  the  power  is  cut  off. 
The  driving  force  is  preferably  a  jet  of  water  acting  on  a 
turbine  arrangement  attached  to  the  base  of  the  spindle  of 
the  plate,  but  this  is  only  available  when  a  sufficient  pressure 
of  water  (40  to  50  lbs.  to  the  square  inch  if  a  single  jet  is 
used  or  20  to  30  lbs.  with  a  double  jet)  can  be  obtained. 
When  electricity  is  used  as  the  driving  force,  it  is  best  applied 
by  a  motor  and  band  working  on  a  collar  attached  to  the 
spindle,  and  capable  of  running  free  when  the  motor  is 
stopped.  The  speed  of  the  motor  must  be  regulated  by  a 
series  of  stops  connected  with  the  starting  lever.  This  type 
of  instrument,  as  supplied  by  Messrs.  Maw,  Son  and  Thompson, 
is  depicted  in  the  illustration. 

To  perform  the  reaction. — Make  a  mark  on  a  Wright's 
pipette  about  1  inch  from  the  tip.     Draw  up  to  the  mark  a 


BLOOD    SERUM— AGGLUTININS   AND   OPSONINS    61 

volume  of  the  washed  cells,  and  admit  a  column  of  air.  Draw  up 
a  volume  of  the  normal  serum,  and  admit  a  column  of  air.  Draw 
up  a  volume  of  the  bacterial  suspension.  Blow  out  the  con- 
tents of  the  pipette  into  a  clean  watch  glass  and  mix  them 
thoroughly.  Draw  up  the  mixture  into  the  pipette,  seal  the 
end  of  the  pipette  in  the  flame,  and  remove  the  rubber  teat. 
Make  a  note  of  the  time,  and  place  the  pipette  in  the  incubator. 
Eepeat  the  process,  substituting  the  patient's  for  the  normal 
serum.  At  the  end  of  20  minutes  remove  each  pipette  from 
the  incubator,  break  off  the  end,  slip  on  the  teat,  and  blow 
the  contents  on  to  a  clean  slide.  With  a  second  slide  make 
a  thick  film  on  the  first  slide  as  if  making  a  blood  film.  Dry 
the  film  by  rapidly  waving  in  the  air.  Dip  the  film  when 
dry  in  a  beaker  of   tap  water  and   keep   it  there  until  the 


Fig.  9. — Centrifugal  Machine. 


haemoglobin  is  dissolved  out  of  the  red  cells.  Without  allow- 
ing to  dry,  stain  the  film.  The  stain  employed  depends  upon 
the  bacteria  present  in  the  suspension.  In  the  case  of  bacteria 
which  take  the  ordinary  dyes,  filter  carbol  thionin  on  to  the 
slide  and  leave  for  3  minutes.  Wash  in  tap  water  and 
blot  dry.  In  the  case  of  tubercle  bacilli,  stain  for  5  minutes 
in  hot  filtered  carbol  fuchsin.  Decolorise  in  12  per  cent, 
nitric  acid.  Wash  in  tap  water.  Counterstain  for  2  minutes 
with  dilute  methylene  blue.  Wash  in  water  and  blot  dry. 
For  counting  the  films,  use  the  oil  immersion  lens,  and 
choose  the  thicker  parts  of  the  films,  preferably  towards 
the  edges  of  the  slides.  Count  50  or  100  polynuclear 
leucocytes  and  the  number  of  organisms  contained  in  them. 
To  estimate  the  opsonic  index  divide  the  number  of  organisms 
counted  in  the  slide  made  from,  the  tube  which  contained  the 
patient's    serum    by  the    number    of    organisms   counted  in 


62  CLINICAL   PATHOLOGY. 

the  same  number  of  cells  on  the  control  or  normal  slide. 
Thus,  if  100  phagocytes  in  the  pathological  mixture  contained 
300  organisms  and  in  the  normal  mixture  200,  the  opsonic 
index  of  the  patient's  serum  is  1*5. 

The  number  of  organisms  per  phagocyte  depends  largely 
on  the  strength  of  the  bacterial  suspension,  and  the  ideal 
number  ingested  is  an  average  of  about  3  per  cell.  If  the 
number  of  bacteria  taken  up  falls  much  below  the  proper 
standard,  or  if  the  cells  are  over  full  and  many  of  them  contain 
clumps  of  organisms,  the  experiment  must  be  repeated. 

The  value  of  the  opsonic  index. — The  claims  of  this 
reaction  to  a  place  in  clinical  medicine  are  two  in  number. 
It  has  been  said  that  the  dosage  of  vaccines  should  be  regulated 
by  frequent  estimations  of  the  opsonic  index,  on  the  grounds 
that  a  dose  given  with  a  falling  index  may  depress  the  index 
still  further  and  do  harm,  and  that  the  nature  of  the  response 
on  the  part  of  the  index  is  an  indication  of  the  suitability  in 
size  of  the  dose.  It  has  been  further  claimed  that  variations 
of  the  opsonic  index  either  above  or  below  the  normal  to  any 
organism  is  evidence  of  infection  by  that  organism.  The 
opsonic  index  has  thus  been  used  both  in  the  treatment  and 
in  the  diagnosis  of  disease.  Considerable  controversy  has 
ranged  around  the  value  of  this  test,  and  the  opinion  of  the 
majority  at  the  present  would  seem  to  be  that,  whatever  guide 
the  opsonic  index  may  have  been  in  the  past  as  a  control  of 
vaccine  treatment,  it  is  no  longer  necessary  to  rely  upon  it, 
and  that  as  a  method  of  diagnosis,  the  variations  in  the  index 
being  on  the  whole  less  than  the  variations  arising  from  the 
errors  of  technique,  little  dependence  is  to  be  placed  upon  it. 
The  errors  in  the  index  due  solely  to  the  technique  are  no 
doubt  considerable,  and  the  whole  process  is  a  very  artificial 
one,  which  estimates  not  the  immunity  of  the  body  to  a  parti- 
cular infection,  but  a  single  immune  process,  the  serum 
opsonin.  Further,  the  resulting  index  can  only  be  approxi- 
mate, since  in  counting  from  50  to  100  cells  which  contain  a 
number  of  organisms  ranging  from  none  at  all  to  nearly  20, 
considerable  variations  must  occur  while  enumerating  different 
series  of  cells  in  the  same  slide.  The  experimental  error  is 
probably  at  least  25  per  cent.,  or  barely  within  the  range  of 
variations  met  with  in  disease.  The  method  is  described  here 
partly  because  it  has  been  very  widely  used  and  is  still  used 


BLOOD    SERUM— AGGLUTININS   AND   OPSONINS    63 

to  some  extent,  but  mainly  because  the  conception  of  the 
opsonic  index  is  acknowledged  to  have  been  a  fine  one  and 
the  technique  of  the  method  is  instructive  and  of  great  value 
in  experimental  work. 

Modifications  of  the  opsonic  index. — Attempts  have 
been  made  to  lower  the  experimental  errors  of  the  reaction  by 
modifications  of  the  actual  technique,  and  it  may  be  said  that, 
except  for  minor  alterations  adopted  by  individual  workers, 
no  technical  changes  of  importance  have  been  introduced  into 
Wright's  original  method.  More  important  alterations  have 
been  suggested  in  the  theory  and  working  of  the  reaction.  It 
has  been  claimed  that  in  localised,  and  particularly  in  tuber- 
culous, affections  massage  or  other  exercise  of  the  affected 
part  leads  to  a  great  increase  of  opsonin  in  the  general  circu- 
lation. For  purposes  of  diagnosis  the  index  is  taken  with  the 
part  at  rest,  and  again  after  exercise.  Any  considerable 
fluctuations  in  the  index  are  considered  as  evidence  of  infection. 
Other  observers  heat  the  serum  with  the  view  of  destroying 
the  general  opsonin  and  leaving  the  more  thermo-stable  specific 
opsonin ;  and  it  is  claimed  that  with  heated  sera  more 
difference  can  be  demonstrated  between  a  normal  and  an 
immune  serum.  The  amount  of  phagocytosis  induced  by 
these  heated  sera  is,  however,  small.  Another  modification  is 
that  known  as  the  hsemophagocytic  index.  This  method  is 
simple  to  perform,  and  has  the  additional  advantage  of  esti- 
mating the  net  result  of  alterations  in  the  activity  of  the 
leucocytes  as  well  as  in  the  amount  of  opsonin  in  the  serum. 
It  was  originally  claimed  that  the  phagocytes  played  a  purely 
secondary  part  and  did  not  differ  in  health  or  in  disease,  but 
this  has  been  shown  to  be  erroneous.  The  phagocytes  in 
disease  may  be  much  more  active  than  in  health,  or  they  may 
be  less  active ;  nor  does  their  activity  vary  with  the  amount  of 
opsonin  in  the  serum.  To  estimate  the  haemophagocytic 
index  it  is  necessary  to  make  the  bacterial  suspension  with 
citrated  salt  solution  instead  of  with  normal  saline.  A  volume 
of  the  patient's  blood  is  drawn  direct  from  the  finger  into  a 
pipette,  and  a  volume  of  the  bacterial  suspension  is  mixed, 
and  incubated  with  it.  The  control  normal  blood  is  treated 
in  the  same  way.  After  incubation  the  slides  are  made  and 
counted  as  in  the  ordinary  method.  In  this  method  the 
patient's  own  phagocytes  act  upon  the  opsonised  bacteria,  and 


64  CLINICAL   PATHOLOGY. 

the  blood  is  subjected  to  little  mechanical  disturbance.  It 
cannot  be  said  that  any  of  the  above  modifications  have 
rendered  the  estimation  of  the  opsonic  index  of  any  great 
practical  value.  On  theoretical  grounds  the  hsemophagocytic 
index  has  some  advantage  in  that  it  is  a  measure  of  the 
phagocytic  power  of  the  blood,  not  merely  of  the  amount  of 
opsonin,  and  in  practice  it  is  readily  obtained. 


CHAPTER    V. 

THE    BLOOD    SERUM  (continued) COMPLEMENT    FIXATION    TESTS 

THE    WASSERMANN    REACTION. 

The  Wassermann  Reaction. — This  reaction  was  devised 
as  a  test  for  the  presence  in  the  blood  of  a  syphilitic 
patient  of  the  specific  antibody  to  the  toxin  of  the  Spirochceta 
pallida.  The  nature  and  meaning  of  the  reaction  are  most 
readily  explained  by  a  brief  account  of  the  steps  in  our 
knowledge  of  immunity  which  led  to  its  discovery.  It  was 
shown  by  Bordet  that  if  an  animal  of  one  species,  for  example 
a  guinea-pig,  were  injected  several  times  with  the  red  cells 
of  an  animal  of  a  different  species,  for  example  a  rabbit,  then 
the  blood  serum  of  the  guinea-pig  acquired  in  very  high 
degree  the  property  of  hsemolysing,  or  dissolving  the  haemo- 
globin out  of,  the  red  cells  of  the  rabbit  and  of  no  other  species. 
That  is  to  say  :  — 

Immunised  guinea-pig's  serum  -f-  normal  rabbit's  red  cells 
=  haemolysis. 

It  has  been  shown  also  that  the  sera  of  some  animals  are 
capable  under  normal  conditions  of  haemolysing  the  red  cells 
of  other  widely  different  species.  For  example,  human  serum 
haemolyses  sheep's  red  cells.  Such  a  haemolysing  serum  is  a 
normal  or  natural  haemolytic  serum  as  opposed  to  the  serum 
of  the  injected  guinea-pig,  which  is  an  immune  serum.  A 
natural  serum  is  never  so  powerfully  hemolytic  as  an  immune 
one. 

It  was  next  shown  that  if  the  immune  serum  were  previously 
heated  to  60°  C.  and  then  added  to  the  red  cells  no  haemolysis 
occurred : — 

Heated  immune  serum  (guinea-pig)  +  red   cells  (rabbit) 
=  no  haemolysis. 

It  was  found  that  if  a  very  small  quantity  of  normal  guinea- 
pig's  serum,  which  of  itself  had  no  haemolytic  effect,  were 
added  to  the  above  mixture  haemolysis  then  took  place  : — 

Heated   immune    serum  (guinea-pig)  +  normal    serum 
(guinea-pig)  +  red  cells  (rabbit)  =  haemolysis. 

p.  5 


66  CLINICAL   PATHOLOGY. 

The  explanation  of  this  apparent  anomaly  is  that  the  hemo- 
lytic substance  or  hemolysin  present  in  the  immune  serum 
consists  of  two  bodies,  both  of  which  are  necessary  for  the 
reaction.  One  body,  the  complement,  is  destroyed  by  heat,  is 
present  in  any  serum  and  is  non-specific.  The  other  body,  the 
amboceptor,  is  not  affected  by  moderate  heat,  is  present  only 
in  immune  sera  (and  in  a  natural  hemolytic  serum),  and  is 
specific.  The  hemolytic  action  depends  upon  a  union  of  red 
cell  with  amboceptor,  and  then  a  further  linking  of  red  cell  and 
amboceptor  with  complement.  Bed  cells  to  which  a  heated 
immune  serum  has  been  added  combine  with  the  amboceptor 
present,  but  are  not  evidently  affected.  Such  red  cells  are 
known  as  sensitised  red  cells,  since  they  only  need  the 
addition  of  complement  for  hemolysis  to  take  place. 

It  was  also  discovered  that  what  is  true  of  hemolysins  is 
true  of  toxins  and  other  similar  bodies,  and  further  that  any 
substance  of  sufficient  chemical  complexity  inoculated  into  an 
animal  leads  to  the  appearance  in  the  animal's  blood  of  a  body 
of  the  nature  of  an  amboceptor  which  is  capable  of  uniting 
chemically  with  the  substance  inoculated.  If  the  substance 
inoculated  be  a  toxin  the  combining  substance  produced  is 
called  an  antitoxin,  and  if  a  bacillus  be  inoculated  a  bacterio- 
lysin  is  produced.  Any  substance  capable  of  producing  an 
antibody  is  known  as  an  antigen ;  thus  in  the  case  of  a  hemo- 
lytic serum  the  antigen  is  a  red  cell. 

Bordet  and  Gengou  then  showed  that  the  antibody  in  the 
serum  of  an  inoculated  animal,  brought  into  contact  with  the 
specific  antigen  in  the  presence  of  complement,  united  with 
antigen  and  complement.  They  proved  that  this  triple  com- 
bination had  taken  place  by  adding  the  mixture  to  sensitised 
red  cells  and  demonstrating  that  no  hemolysis  of  the  red  cells 
occurred  owing  to  the  previous  fixation  of  complement.  The 
reaction  was  found  to  take  place  in  numerous  infections  and 
to  be  capable  of  being  applied  as  a  method  of  diagnosis  in 
human  pathology.  They  found,  for  example,  that  if  the  serum 
of  a  patient  convalescent  from  typhoid  fever  were  heated  (to 
destroy  the  complement,  but  not  the  antibody)  and  incubated 
with  typhoid  bacilli  (antigen)  and  guinea-pig's  serum  (com- 
plement), and  then  added  to  sensitised  red  cells,  no  hemo- 
lysis took  place,  because  typhoid  bacilli,  typhoid  antigen 
and  complement  had  united  and  no  complement  remained  for 


BLOOD  SERUM— COMPLEMENT  FIXATION  TESTS.   67 

the  sensitised  red  cells  ;  whereas  if  normal  human  serum 
(heated)  were  incubated  with  typhoid  bacilli  and  guinea-pig's 
serum  and  then  added  to  the  red  cells,  haemolysis  took  place 
readily,  since  normal  serum  contains  no  antibody  to  the 
typhoid  bacillus  and  has  therefore  no  substance  capable  of 
uniting  with  the  bacilli  and  the  complement  in  the  guinea-pig's 
serum.  Consequently  complement  is  available  to  combine 
with  the  sensitised  red  cells  and  hemolyse  them.  This 
reaction  is  known  as  the  Bordet-Gengou  reaction,  or  the  com- 
plement deviation  test.  It  is  a  specific  test.  Antibody  can 
unite  only  with  its  specific  antigen.  Specific  antigen  and 
specific  antibody  must  both  be  present  before  any  combination 
with  the  (non-specific)  complement  can  take  place. 

The  original  Wassermann  reaction  was  simply  an  applica- 
tion of  the  Bordet-Gengou  test  to  the  diagnosis  of  syphilis.  In 
the  case  of  syphilis  the  specific  antigen  is  the  Spirochceta 
pallida,  and,  since  this  organism  cannot  be  cultivated  on  any 
of  the  ordinary  media,  Wassermann  conceived  the  idea  of 
utilising  some  organic  extract  rich  in  spirochetes,  and  for  this 
purpose  made  use  of  an  extract  of  the  liver  of  a  syphilitic 
foetus.  The  reaction  was  completely  successful,  since  it  was 
found  that  the  heated  serum  of  a  syphilitic  patient  incubated 
with  guinea-pig's  complement  and  the  extract  of  syphilitic 
antigen  absorbed,  by  virtue  of  the  syphilitic  antibody,  the 
complement  from  the  mixture,  whereas  normal  serum  failed 
to  do  so.  The  test  appeared  to  afford  a  specific  proof  of 
immunity  to  the  spirochete. 

The  meaning  of  the  test  is  so  far  clear.  It  has,  however, 
since  been  demonstrated  that  the  Wassermann  reaction  is 
not  an  essential  combination  between  syphilitic  antigen  and 
antibody,  since  the  spirochete  can  be  altogether  omitted  from 
the  mixture.  If  an  alcoholic  extract  of  human  heart  muscle  or 
even  of  guinea-pig  heart  muscle  be  substituted  for  the  syphilitic 
liver  extract,  the  reaction  is  equally  reliable  in  as  much  as 
it  is  obtained  with  syphilitic  and  not  with  normal  sera.  The 
essential  substances  in  the  extract  employed  as  antigen  are 
found  to  be  certain  fatty  bodies,  or  lipoids,  such  as  can  be 
extracted  from  normal  organs.  Why  lipoids  are  capable  of 
acting  as  syphilitic  antigen  in  place  of  the  true  antigen,  the 
spirochete,  is  unexplained.  It  has  been  suggested  that  the 
syphilitic   virus   has   an   affinity   for   the    body   lipoids   and 

5—2 


68  CLINICAL   PATHOLOGY. 

combines  with  them  forming  a  toxo -lipoid,  and  that  an 
antibody  is  produced  to  the  toxo-lipoid  which  would  be  capable 
of  combining  with  it  or  with  lipoid.  According  to  this 
explanation  the  reaction  is  a  chemical  combination  between 
anti-toxo-lipoid  present  in  syphilitic  serum,  lipoid  acting  as 
antigen  in  place  of  the  toxo-lipoid,  and  complement.  (It  may 
be  added  that  the  toxophore  group  of  a  toxin  is  not  essential 
for  the  combination  of  toxin  and  antitoxin,  and  that  anti- 
diphtheritic  toxin  can  combine  with  the  diphtheria  toxoid  or 
non-poisonous  toxin.) 

It  has  also  been  suggested  that  syphilitic  serum  contains 
some  abnormal  proteid  derived  from  the  destructive  action  of 
the  spirochete  on  proteo-lipoid  compounds  in  the  body,  which 
proteid  when  added  to  lipoid  outside  the  body  is  precipitated 
and  in  its  precipitation  carries  down  complement.  On  this 
theory  the  removal  of  complement  from  the  Wassermann 
mixture  is  a  mechanical  rather  than  a  purely  chemical  action. 

"Whatever  may  be  the  explanation  of  the  reaction,  it  follows 
that,  since  the  spirochete  may  be  replaced  as  antigen  by  lipoid, 
a  positive  reaction  is  not  evidence  of  a  specific  immunity  to 
the  Spirochata  pallida,  but  rather  of  a  peculiar  and  abnormal 
lipoid  metabolism  such  as  might  be  common  to  more  than  one 
disease.  It  is  further  probable  that  a  positive  Wassermann 
reaction  means  not  so  much  that  a  person  is  immune  to 
syphilis  as  that  he  is  still  infected  by  syphilis. 

The  reaction  in  diagnosis, — Since  the  reaction  is  not  a 
truly  specific  one  we  might  expect  it  to  be  present  in  some 
diseases  other  than  syphilis,  and  this  is  found  to  be  the  case. 
A  positive  reaction  is  associated  with  certain  diseases  foreign 
to  this  country,  including  sleeping  sickness,  yaws,  and  some 
forms  of  leprosy.  A  positive  reaction  is  also  found  occasionally, 
and  for  a  brief  period  only,  in  scarlet  fever.  "With  these 
exceptions — and  from  the  point  of  view  of  diagnosis  they  are 
unimportant  exceptions — a  positive  Wassermann  reaction  is 
very  definite  evidence  of  a  syphilitic  affection.  As  to  the 
occurrence  of  the  reaction  in  the  various  stages  of  syphilis  : — 

In  primary  syphilis  the  reaction  is  nearly  always  positive, 
and  becomes  so  at  periods  varying  from  4  to  12  weeks  after 
infection.  A  negative  reaction  at  any  date  later  than  this  in 
the  case  of  a  doubtful  sore  is  strongly  opposed  to  the  diagnosis 
of  syphilis.     Since  it  is  of  the  utmost  importance,  however, 


BLOOD  SERUM— COMPLEMENT  FIXATION  TESTS.   69 

to  commence  treatment  at  the  earliest  possible  date,  and 
preferably  before  the  reaction  has  become  positive,  there  is 
fortunately  no  necessity  to  rely  upon  the  Wassermann  test. 
In  cases  of  any  doubt  the  presence  of  the  specific  spirochsete 
is  conclusive  (page  142). 

In  secondary  syphilis  if  untreated  the  test  is  practically 
always  positive,  and  a  negative  reaction  almost  contra- 
indicates  syphilis. 

In  tertiary  syphilis  positive  reactions  are  not  quite  so 
invariable ;  the  test  is,  however,  positive  in  from  80  to  90  per 
cent,  of  treated  and  untreated  cases.  It  is  perhaps  more 
common  to  find  a  positive  reaction  in  some  tertiary  lesions 
than  in  others ;  a  negative  reaction  with  a  thoracic  aneurysm, 
for  example,  is  extremely  rare. 

In  latent  syphilis,  that  is  to  say  in  cases. of  past  infection 
with  no  present  manifestations  of  the  disease,  the  reaction 
is  positive  in  only  from  30  to  40  per  cent.  This  class 
includes  those  who  have  been  cured,  and  it  is  probable  that  a 
positive  reaction  in  a  person  who  has  had  syphilis  some  years 
previously  is  evidence  that  he  is  still  liable  to  recurrence  of 
the  disease,  and  that  a  negative  reaction  indicates  that  he  has 
been  cured. 

In  parasyphilis  the  reaction  is  positive  in  almost  every 
case  of  general  paralysis,  but  in  only  from  60  to  70  per  cent, 
of  tabetics.  The  serum  test  is  of  great  value  in  the  diagnosis 
of  syphilis  of  the  central  nervous  system  and  of  parasyphilis ; 
it  does  not,  however,  help  us  to  distinguish  between  these 
conditions.  Further  information  is  obtained  by  examination 
of  the  cerebro-spinal  fluid  (page  203).  In  both  syphilis  of  the 
central  nervous  system  and  parasyphilis  a  lymphocytosis  is 
present  in  the  fluid ;  but  in  syphilis  the  Wassermann  test  of 
the  spinal  fluid  is  negative  as  a  rule,  while  in  parasyphilis  it 
is  nearly  always  positive  and,  particularly  in  general  paralysis, 
very  strongly  positive.  In  other  forms  of  syphilis  there  is  no 
alteration  in  the  spinal  fluid. 

In  congenital  syphilis  the  reaction  is  practically  always 
strongly  positive. 

The  Wassermann  test  is  thus  seen  to  be  of  great  value 
in  the  diagnosis  of  all  syphilitic  lesions ;  but  it  must  be 
remembered  that  a  negative  reaction  sometimes  occurs  with 
lesions  undoubtedly  syphilitic,  and  that  a  positive  reaction 


70  CLINICAL  PATHOLOGY. 

means  that  a  patient  is  tainted  with  syphilis — it  does  not 
necessarily  mean  that  the  particular  lesion  for  which  he  is 
under  observation  is  syphilitic.  In  the  case  of  a  doubtful 
tumour,  for  example,  a  negative  reaction  is  evidence  against 
syphilis ;  a  positive  reaction  yields  the  valuable  information 
that  the  patient  has  had  syphilis  and  is  probably  still  infected, 
but  it  does  not  tell  us  that  the  "  tumour  "  is  a  gumma. 

It  may  be  added  that  we  are  not  dependent  upon  the 
Wassermann  test  for  our  diagnosis  of  all  cases  of  syphilis. 
The  majority  of  syphilitic  lesions  are  sufficiently  obvious  on 
clinical  grounds  to  make  their  recognition  certain,  particularly 
in  the  presence  of  a  truthful  history.  The  student  at  a 
hospital  sees  the  reaction  tested  in  many  cases  as  a  matter  of 
routine,  and  is  in  danger  of  falling  into  the  error  of  placing 
too  much  reliance  on  a  test  and  neglecting  the  cultivation  of 
his  clinical  experience. 

The  response  of  the  reaction  to  treatment. — The 
alterations  which  may  take  place  in  the  reaction  as  the  result 
of  treatment  depend  partly  upon  the  efficiency  of  the 
treatment  and  partly  upon  the  stage  of  the  disease  at  which 
treatment  is  being  undertaken.  In  the  primary  and 
secondary  stages  of  the  disease  the  reaction  alters  rapidly 
in  response  to  energetic  treatment,  and  should  become 
negative  in  from  6  to  12  weeks.  A  positive  reaction  becomes 
negative  most  rapidly  after  intravenous  injections  of 
salvarsan,  and  readily  also  after  intensive  mercurial  treat- 
ment. During  the  treatment  the  reaction  may  be  seen  to 
change  from  strongly  positive  to  partial  and  finally  to 
negative.  A  negative  reaction  at  this  stage  does  not  mean 
that  the  patient  is  cured  unless  it  remains  negative  for  many 
months,  and  possibly  not  even  then,  since  if  further  treatment 
is  omitted  a  negative  reaction  may  again  become  positive  and 
be  followed  by  a  relapse  in  the  symptoms  and  physical  signs. 
If  the  treatment  is  inadequate  during  the  primary  and 
secondary  changes  the  reaction  remains  positive  or  becomes 
partial  only,  and,  since  there  can  be  little  question  that  a 
permanently  negative  reaction  is  the  goal  to  be  aimed  at,  the 
effect  of  treatment  is  reasonably  controlled  by  the  state  of  the 
reaction,  which  appears  to  be  a  more  sensitive  guide  than  the 
clinical  condition  of  the  patient.  In  the  tertiary  stage  of 
syphilis  the  behaviour  of  the  reaction  is  quite  different,  since 


BLOOD  SERUM— COMPLEMENT  FIXATION  TESTS.  71 

at  this  stage,  whatever  the  treatment  adopted,  it  is  quite 
exceptional  to  obtain  a  completely  negative  reaction,  and 
frequently  very  little  change  is  observable  in  it.  The 
behaviour  of  the  reaction  in  this  stage  of  syphilis  is  compar- 
able with  the  effect  of  treatment  in  the  clinical  condition  of 
the  patient,  since  it  is  notoriously  difficult  to  cure  the  disease 
after  tertiary  symptoms  have  manifested  themselves.  In  the 
tertiary  stage  lesions  frequently  clear  up  rapidly  under 
treatment,  but  have  the  greatest  tendency  to  recur,  and  a  very 
small  percentage  of  cases  is  actually  cured. 

The  Wassermann  reaction  is  thus  a  valuable  guide,  not  only 
in  the  diagnosis  of  syphilis,  but  also  in  estimating  the  effect  of 
treatment.  The  value  of  the  reaction  in  prognosis  is  less 
certain.  It  is  probable  that  a  patient  who  has  had  syphilis, 
and  whose  reaction  is  found  to  be  negative  a  year  or  more 
after  treatment  has  been  stopped,  is  cured  of  the  disease.  It  is 
probable  also  that  patients  who  have  had  no  symptoms  for 
some  years  but  are  found  to  still  have  a  positive  reaction, 
are  liable  to  tertiary  manifestations  or  to  parasyphilis.  There 
is  at  present,  however,  no  statistical  proof  of  these  statements, 
since  the  reaction  has  not  been  available  for  a  sufficient 
period. 

The  technique  of  the  Wassermann  reaction. — There 
are  numerous  methods  of  performing  the  reaction,  and  they 
differ  very  considerably  from  one  another.  Two  methods  are 
described  here,  and  both  are  widely  used  in  this  country.  The 
first  method  is  given  because  it  more  closely  corresponds 
to  the  original  reaction  of  Wassermann,  the  second  because  it 
is  a  more  simple  method  and  at  the  same  time  a  very 
sensitive  one.  The  advantages  and  disadvantages  of  the  two 
methods  will  be  discussed  at  the  end  of  the  chapter. 

Method  1. — A  modification  of  the  original  technique. — The 
materials  required  are  serum  tubes,  small  test  tubes,  a 
graduated  pipette  (0*1  to  1  c.c),  watch  glasses,  saline,  a 
centrifugal  machine,  etc.,  and  in  addition  fresh  guinea-pig's 
serum  (complement),  immune  serum  (rabbit  to  sheep),  red 
cells  (sheep),  an  alcoholic  extract  of  human  heart  muscle 
(antigen),  human  sera  (normal,  syphilitic,  unknown). 

The  complement  is  fresh  guinea-pig:s  serum.  It  is 
obtained  by  plunging  a  needle  attached  to  a  5  c.c.  syringe  into 
the  animal's  heart,  withdrawing  the  blood  and  ejecting  it  into  a 


72  CLINICAL   PATHOLOGY. 

centrifuge  tube.  After  centrifuging  the  clear  serum  is  pipetted 
off,  and  should  be  used  the  same  day  or  within  24  hours. 
The  guinea-pig  is  usually  none  the  worse  for  the  operation. 

The  immune  serum  is  the  serum  of  an  animal  which 
has  been  inoculated  with  washed  sheep's  corpuscles.  The 
sheep's  blood  is  obtained  from  the  butcher  and  received  into 
sterile  citrated  salt  solution.  It  is  then  centrifuged  and 
washed  several  times  with  saline.  About  2  c.c.  of  the  washed 
corpuscles  are  injected  beneath  the  skin  of  a  rabbit,  and  the 
injection  is  repeated  every  7  days  for  3  or  4  injections  or  until 
a  serum  is  obtained  strongly  hemolytic  for  sheep's  cells.  It 
is  somewhat  a  matter  of  chance  when  a  serum  becomes 
strongly  immune.  The  rabbit's  blood  is  removed  by  a 
needle  and  syringe  from  a  vein  in  the  ear,  and  after  standing 
is  centrifuged.  The  clear  serum  is  pipetted  off  into  sterile 
capsules.  The  capsules  are  sealed  and  heated  in  water  at 
60°  C.  for  15  minutes.  They  are  then  stored,  and  will  keep 
their  specific  attributes  for  several  months. 

The  red  cells  are  those  of  a  sheep ,  and  are  obtained  and 
treated  in  the  same  manner  as  those  used  for  injection  into  a 
rabbit.  A  5  per  cent,  saline  suspension  of  the  final  washed 
deposit  is  used  for  the  reaction. 

The  above  materials — namely,  the  complement,  the  ambo- 
ceptor of  the  heated  immune  serum,  and  the  red  cells — 
constitute  the  hemolytic  system. 

The  antigen  is  prepared  as  follows  :  A  heart  is  obtained 
from  any  cadaver  in  the  post-mortem  room,  and  slices  of  the 
muscular  portions  are  removed  with  a  clean  knife,  blotted  dry, 
and  weighed.  The  slices  are  transferred  to  a  mortar  and 
thoroughly  ground  up  with  absolute  alcohol  in  the  proportion 
of  1  gramme  of  heart  muscle  to  5  c.c.  of  alcohol.  The  mixture 
is  transferred  to  a  sterile  test  tube  and  heated  in  a  water  bath 
at  60°  C.  for  1  hour  and  then  incubated  for  24  hours  at  37°  C. 
The  mixture  is  then  filtered  through  an  ordinary  filter  paper 
into  a  sterile  bottle  and  stored  in  a  closed  cupboard  at  room 
temperature.  Extracts  obtained  in  this  way  are  extremely 
constant  in  strength,  and  will  keep  without  variation  for  some 
months.  They  should,  however,  be  standardised  before  use 
and  be  constantly  controlled. 

The  human  sera  are  obtained  in  the  same  way  as  for  the 
Widal  reaction   (page  51).     Two  tubes,  at   least   hatf -filled, 


BLOOD  SERUM— COMPLEMENT  FIXATION  TESTS.  73 

should  be  obtained  from  each  case,  and  any  tube  which  shows 
haemolysis  in  the  serum  after  centrifuging  must  be  discarded. 
The  serum  is  pipetted  off  into  capsules  and  inactivated  by 
heat  in  the  same  manner  as  the  immune  serum.  Serum  is 
required  from  a  non-syphilitic  source,  from  a  known  case  of 
syphilis,  and  from  the  cases  to  be  tested. 

The  salt  solution  is  made  in  the  usual  manner  from  pure 
sodium  chloride  and  distilled  water.  For  the  purposes  of  this 
reaction  it  should  be  freshly  prepared  and  of  a  strength  of 
exactly  0'9  per  cent. 

Standardisation  of  the  materials. — The  most  variable 
reagent  is  the  immune  serum,  and  this  is  standardised  as 
follows  :  A  1  in  1,000  dilution  of  the  serum  is  made  with 
saline  and  into  a  series  of  tubes  "1,  *2  up  to  *9  c.c.  of  this 
diluted  serum  are  put,  and  each  tube  is  made  up  to  "9  c.c. 
with  saline.  To  each  tube  is  added  "1  c.c.  of  a  1  in  4  dilution 
of  guinea-pig's  serum  and  *5  c.c.  of  the  5  per  cent,  suspension 
of  sheep's  red  cells.  The  tubes  are  incubated  for  1  hour  at 
37°  C.  and  then  allowed  to  stand  aside  for  the  corpuscles  to 
settle.  A  "  unit  "  of  amboceptor  was  present  in  that  tube  which 
contained  the  smallest  quantity  of  immune  serum  necessary  for 
complete  haemolysis.  Two  and  a  half  units  are  used  for  the 
Wassermann  reaction.  The  standard  of  the  immune  serum 
having  been  fixed,  it  is  usual  on  each  occasion  to  fix  the 
standard  of  the  complement,  and  this  is  done  in  exactly  the 
same  way,  except  that  a  fixed  quantity  (2^  units)  of  amboceptor 
is  added  to  each  tube  and  the  amount  of  complement  is  varied. 
Two  units  of  complement  are  used  for  the  test,  and  this 
amount  is  usually  contained  in  *025  c.c.  of  serum  or  "1  c.c.  of 
a  1  in  4  dilution.  The  amount  of  complement  present  in  the 
serum  is  extremely  constant,  and  it  is  almost  unnecessary  to 
estimate  it. 

The  antigen  is  standardised  as  follows :  A  series  of 
dilutions  of  the  alcoholic  extract  are  made  in  saline,  and 
•025  c.c.  of  complement  added  to  each  tube.  The  tubes  are 
shaken  and  incubated  for  1  hour;  then  to  each  tube  are 
added  2J  units  of  amboceptor  and  -5  c.c.  of  the  diluted  red 
cells.  The  tubes  are  again  shaken  and  incubated  for  1  hour. 
The  smallest  amount  of  antigen  is  noted  which  has  been  able 
of  itself  to  absorb  the  complement,  and  one-third  of  this 
amount  is  used  in  the  reaction. 


74  CLINICAL   PATHOLOGY. 

To  perform  the  reaction  prepare  a  series  of  tubes  in 
pairs,  sufficient  for  2  for  each  serum  to  be  examined,  2  for  the 
normal  serum,  2  for  the  syphilitic  serum,  as  well  as  3  control 
tubes,  which  are  to  contain  no  human  serum.  Sufficient 
saline  is  placed  in  each  tube  to  make  the  final  volumes  equal 
(i.e.  from  0*7  to  1*0  c.c.  of  saline).  Into  one  of  each  pair  of 
tubes  place  the  requisite  quantity  of  antigen  as  well  as  into  the 
first  of  the  3  control  tubes.  Into  every  tube  except  the  last 
control  tube  place  "025  c.c.  of  complement  (this  is  conveniently 
diluted  with  saline  to  make  up  *1  c.c.  of  fluid).  Into  each  pair 
of  tubes  place  "1  c.c.  of  the  appropriate  serum  (i.e.  the 
inactivated  human  serum).  After  mixing  incubate  1  hour  at 
37°  C.  After  incubation  add  to  all  the  tubes  '5  c.c.  of  the 
sheep's  red  cells  and  2J  units  of  the  amboceptor.  Again  shake 
and  incubate  for  1  hour  at  37°  C,  then  stand  the  tubes  on  ice 
for  the  corpuscles  to  settle. 

The  3  control  tubes  should  show  complete  haemolysis  in  the 
two  containing  complement  and  none  in  the  third  tube. 

The  normal  pair  of  tubes  should  show  haemolysis  in  both 
tubes.  The  pair  of  tubes  containing  syphilitic  serum  should 
show  haemoly sis  in  the  tube  with  no  antigen  and  no  haemolysis 
in  the  tube  with  antigen.  The  tubes  for  the  serum  to  be 
examined  must  show  haemolysis  in  the  tube  without  antigen 
and  either  complete  haemolysis  or  no  haemolysis  in  the  other 
tube,  according  as  the  reaction  is  negative  or  positive. 

The  technique  of  this  test  may  be  further  elaborated  by  a 
quantitative  reaction  instead  of  the  qualitative  one  described 
above.  The  degree  of  positiveness  of  the  test  is  commonly 
determined  by  diluting  the  serum  to  be  tested.  By  using  a 
series  of  dilutions  of  each  serum  the  amount  of  antibody 
present  can  be  estimated  from  the  degree  of  dilution  sufficient 
to  inhibit  haemolysis  in  the  mixture  of  antigen  and  haemolytic 
system. 

In  the  above  technique  each  reagent  of  the  Wassermann 
test  is  treated  separately.  The  antigen  is  obtained  from 
human  heart  muscle ;  the  antibody  is  tested  for  in  the  heated 
human  serum  ;  the  amboceptor  for  the  red  cells  is  present  in 
the  heated  immune  serum ;  the  red  cells  are  those  of  a  sheep  ; 
and  the  complement  is  obtained  from  fresh  guinea-pig's  serum. 
It  may  be  pointed  out  here  that  the  final  mixture  by  this 
method  contains  a  varying  amount  of  immune  body  for  sheep's 


BLOOD   SERUM— COMPLEMENT  FIXATION  TESTS.  75 

cells,  since  human  serum  contains  a  normal  amboceptor  for 
these  cells,  and  is  absent  from  the  3  control  tubes  and  diluted 
when  the  quantitative  test  is  used.  These  variations  are  not 
of  great  importance  provided  sufficient  amboceptor  is  present 
in  the  mixture. 

When  the  test  is  made  on  cerebro-spinal  fluid  that  sub- 
stance is  substituted  for  the  serum,  but  there  is  no  need  to 
heat  the  fluid,  since  it  contains  no  complement. 

Method  2. — The  Hecht-Fleming  modification. — The 
materials  required  are  Wright's  tubes,  serum  tubes,  sheep's 
blood,  antigen,  normal  salt  solution,  human  sera  (normal 
syphilitic  and  unknown),  a  centrifugal  machine,  and  an 
incubator. 

The  sheep's  blood  is  obtained  from  the  butcher,  who  is 
provided  with  a  sterile  glass  bottle  containing  about  30  c.c.  of 
citrated  salt  solution  (0'9  gramme  of  sodium  citrate,  0*9 
gramme  of  sodium  chloride  in  100  c.c.  of  distilled  water)  and 
instructed  to  bleed  the  animal  directly  into  the  bottle,  adding 
about  10  c.c.  of  blood.  The  bottle  is  immediately  inverted 
several  times.  Blood  obtained  in  this  manner  will  keep  for 
several  days  on  ice.  Before  use  a  portion  of  the  mixture  is 
transferred  to  a  centrifuge  tube  and  centrifuged  at  a  moderate 
speed  for  about  3  minutes.  The  supernatant  fluid  is  pipetted 
off,  normal  (0*9  per  cent.)  salt  solution  is  added,  and  the  tube 
is  inverted  several  times.  This  process  is  repeated  four  times — 
or  5  centrifugings  altogether.  The  supernatant  fluid  in  the 
final  washing  must  be  absolutely  clear.  With  a  Wright's 
tube  make  a  10  per  cent,  dilution  of  the  final  deposit  in 
normal  saline. 

The  antigen  consists  of  an  alcoholic  extract  of  human  heart 
muscle  prepared  as  described  under  Method  1.  Before  use 
2  dilutions  of  the  alcoholic  extract,  a  10  per  cent,  and  a  5  per 
cent.,  are  made  with  normal  saline.  In  making  the  dilutions 
the  saline  is  slowly  added  to  the  alcoholic  extract,  and  an 
opalescent  fluid  should  result. 

The  human  sera  are  obtained  in  the  usual  manner,  and 
should  not  be  withdrawn  more  than  24  hours  before  use. 
They  are  unheated,  and  are  proved  to  contain  both  com- 
plement and  amboceptor  for  sheep's  red  cells.  It  is  essential 
in  each  set  of  reactions  to  have  both  a  normal  and  a  syphilitic 
serum  in  order  to  be  certain  that  the  antigen  is  being  used  in 


76  CLINICAL  PATHOLOGY. 

the  required  strength.  The  10  and  5  per  cent,  dilutions  of 
antigen  prepared  as  described  above  are  almost  invariably 
found  capable  of  absorbing  the  complement  completely  from  a 
strongly  positive  syphilitic  serum  and  to  have  little  or  no 
action  on  a  normal  serum.  An  antigen  which  fails  to  act  in 
these  dilutions  should  be  discarded. 

To  perform  the  reaction.— First  stage.— In  a  Wright's 
tube  draw  up  1  volume  of  normal  serum,  4  volumes  of 
normal  saline,  and  1  volume  of  the  10  per  cent,  suspension  of 
sheep's  red  cells.  Mix  thoroughly  on  a  clean  glass  slab.  Draw 
up  the  mixture  into  the  tube  and  seal  the  end  of  the  tube  in 
the  flame.  Remove  the  rubber  teat.  Repeat  the  process  with 
the  syphilitic  serum  and  with  the  unknown  sera,  labelling 
each  tube.  Place  the  tubes  in  the  incubator  at  37°  C.  and 
remove  at  the  end  of  half  an  hour.  Hemolysis  must  be  com- 
plete in  every  tube — that  is  to  say,  the  tube  must  contain  no 
visible  deposit  of  red  cells,  and  the  mixture  must  be  evenly 
tinged  with  haemoglobin.  This  stage  of  the  process  proves 
that  each  serum  contains  a  sufficiency  of  complement  and 
amboceptor  for  the  haemolysis  of  sheep's  red  cells. 

Second  stage. — Draw  up  1  volume  of  normal  serum  and  1 
volume  of  the  10  per  cent,  lipoid.  Mix  thoroughly  on  the  glass 
slab.  Draw  up  the  mixture  into  the  tube  ;  admit  a  consider- 
able column  of  air.  Draw  up  1  volume  of  the  red  cells.  Seal 
the  end  of  the  tube  and  remove  the  teat.  Repeat  the  process 
with  the  5  per  cent,  lipoid.  Put  up  two  similar  tubes  with 
the  syphilitic  serum  and  with  each  of  the  unknown  sera. 
Place  in  the  incubator  at  37°  C.  and  remove  at  the  end  of 
1  hour.  During  this  stage  the  serum  is  in  contact  with  the 
lipoid,  and  union  takes  place  between  complement,  antigen, 
and  syphilitic  antibody  in  those  tubes  which  contain  the 
antibody.  The  amboceptor  for  the  sheep's  red  cells  takes  no 
part  in  the  combination.  The  red  cell  suspension  is  not  in 
contact  with  the  serum  lipoid  mixture,  and  on  removal  from 
the  incubator  it  should  be  noted  that  no  haemolysis  has  taken 
place  in  it. 

Third  stage. — With  a  glass  file  nick  the  end  of  each 
tube  and  break  it  off.  Slip  on  the  teat  and  blow  out  the  con- 
tents of  the  tube.  Mix  thoroughly,  draw  up  again  into  the 
tube,  and  seal  the  end.  Replace  in  the  incubator  for  30 
minutes.     On  removal  from  the  incubator  stand  all  the  tubes 


BLOOD  SERUM— COMPLEMENT  FIXATION  TESTS.   77 

in  a  vertical  position,  preferably  in  the  ice-chest,  until  the 
red  cells  have  settled  to  the  bottom.  Those  tubes  in  which 
haemolysis  has  occurred  show  no  deposit,  or  very  little  deposit, 
of  cells  at  the  bottom,  and  the  supernatant  fluid  is  strongly 
tinged  with  haemoglobin.  These  tubes  are  "  negative."  The 
tubes  in  which  no  haemolysis  has  taken  place  show  a  well- 
marked  deposit  of  red  cells,  and  the  supernatant  fluid  is 
colourless.  These  tubes  are  "  positive."  Other  tubes  may 
show  absence  of  haemolysis  in  the  tube  containing  the  10  per 
cent,  lipoid  and  slight  haemolysis  in  the  5  per  cent.  tube. 
These  tubes  are  "  partial." 

In  this  stage  absence  of  haemolysis  proves  that  complement 
was  removed  from  the  mixture  of  serum  and  antigen  in  the 
previous  stage — in  other  words,  that  the  serum  contained 
syphilitic  antibody.  The  presence  of  haemolysis  proves  the 
presence  of  complement,  and  shows  that  no  antibody  was 
present  in  the  serum  capable  of  combining  with  lipoid  and 
complement  in  stage  2. 

In  performing  the  reaction  with  cerebro-spinal  fluid  the 
first  stage  may  be  omitted,  since  the  fluid  contains  neither 
complement  nor  amboceptor.  In  the  second  stage  take 
1  volume  of  normal  serum,  3  volumes  of  cerebro-spinal 
fluid,  and  1  volume  of  10  per  cent,  lipoid ;  mix  and  draw  up 
separately  1  volume  of  sheep's  cells  as  before.  Repeat  with 
5  per  cent,  lipoid  and  incubate  for  1  hour.  In  this  process 
the  normal  serum  provides  complement  and  amboceptor  and 
the  spinal  fluid,  if  syphilitic,  the  antibody.  The  third  stage 
is  performed  in  the  usual  manner.  A  control  tube  may  be 
put  up  which  contains  saline  in  place  of  the  spinal  fluid. 

If  any  serum  in  stage  1  fails  to  haemolyse  the  sheep's  red 
cells,  the  serum  must  be  heated  to  60°  C.  for  15  minutes 
to  destroy  any  complement  that  may  be  present.  In  stage  2 
1  volume  of  the  heated  serum  is  mixed  with  1  volume  of 
normal  serum  and  1  volume  of  lipoid.  The  remainder  of 
the  test  is  performed  as  above.  One  of  the  objections  made  to 
this  modification  of  the  Wassermann  test  is  that  human  sera 
frequently  fail  to  haemolyse  sheep's  red  cells,  and  some 
observers  have  found  that  from  30  to  40  per  cent,  of  human 
sera  fail  in  this  manner.  Such  findings  arise  from  errors  in 
technique,  of  which  the  most  probable  are  the  keeping  of  the 
sera  more  than  24  hours  before  use  and  the  inadequate  washing 


78  CLINICAL   PATHOLOGY. 

of  the  red  cells.  If  a  comparatively  small  trace  of  sheep's 
serum  is  incubated  with  human  serum  in  the  presence  of 
sheep's  red  cells  no  haemolysis  takes  place,  owing  to  an  inter- 
action between  the  foreign  sera.  If  clue  precautions  are  taken 
to  avoid  these  errors  it  will  be  found  that  only  about  1  per  cent, 
of  human  sera  fails  to  hasmolyse  sheep's  cells. 

If  it  be  necessary  to  use  the  serum  more  than  24  hours  old 
the  serum  should  be  removed  from  the  red  cells  after  centri- 
fuging  and  stored  in  a  capsule  on  ice.  The  serum  may  thus 
remain  active  for  several  days. 

Diluted  Serum  and  Eed  Cells. 


1st  Stage. 
Serum  and  Lipoid.  Eed  Cell  Suspension. 


2nd  Stage. 

Clear  Fluid.  Deposit  of  Eed  Cells. 


3rd  Stage — Positive. 

Complete  Hiemolysis.  No  Deposit. 


3rd  Stage — Negative. 
]?IG.  io. — The  Wright's  Tubes  in  the  Three  Stages  of  the  Eeaction. 

A  comparison  of  the  two  methods.— The  main  advan- 
tage of  the  first  method  is  that  each  component  of  the  reaction 
— the  complement,  the  amboceptor,  and  the  antigen — is 
accurately  measured.  The  disadvantages  are  that  the 
technique  is  involved  and  laborious ;  that  the  human  sera  in 
the  process  of  heating  to  destroy  the  complement  lose  a  part 
of  the  antibody,  and  the  reaction  therefore  loses  in  sensitive- 
ness ;  and  that  the  ultimate  mixture  contains  a  considerable 
variety  of  sera  from  different  animals,  which  interact  to  an 
unknown  extent  and  probably  lead  to  some  loss  of  comple- 
ment on  that  account.  In  practice  there  can  be  no  doubt  of 
the  value  and  accuracy  of  the  method. 


BLOOD  SEEUM— COMPLEMENT  FIXATION  TESTS.   79 

The  main  advantages  of  the  second  method  are  simplicity 
and  the  use  of  unheated  sera.  The  disadvantages  are 
numerous,  but  entirely  theoretical.  It  has  been  objected  that 
the  amount  of  complement  in  human  sera  may  vary,  and 
that  a  positive  serum  with  excess  of  complement  might  give  a 
negative  reaction,  whereas  a  negative  serum  with  a  shortage 
of  complement  might  give  a  positive  result.  There  is  no 
evidence  of  any  such  gross  variations  in  the  amount  of  com- 
plement present  in  fresh  human  sera,  and  the  amount  of 
complement  in  guinea-pig's  serum  is  so  constant  that  it  is 
commonly  not  estimated  by  those  who  prefer  the  original 
method.  In  a  large  number  of  undoubtedly  normal  sera  I  have 
never  seen  a  positive  or  even  partial  reaction.  In  syphilitic 
cases  the  Hecht-Fleming  method  gives  a  higher  percentage  of 
positive  reactions,  and  the  reaction  takes  longer  to  become 
negative  under  treatment  than  with  the  original  method. 

Quantitative  estimations  of  the  reactions  are  most  accurately 
made  by  altering  the  amount  of  the  human  serum,  that  is,  of 
the  syphilitic  antibody,  and  this  is  best  done  by  the  original 
method.  Similar  estimations  can  be  made  by  varying  the 
amount  of  antigen,  as  is. done  in  the  Hecht-Fleming  method, 
and  the  results  are  sufficiently  graded  for  all  clinical  purposes. 

Both  methods  have  their  adherents,  and  both  are  of  great 
value  in  clinical  medicine.  The  second  method  has  been 
subjected  to  considerable  criticism  on  theoretical  grounds,  but 
has  been  found  entirely  satisfactory  in  practice,  and  owing  to 
the  simplicity  of  the  technique  is  described  here  in  full. 

The  complement  fixation  test  in  other  diseases.— 
Before  the  test  described  above  was  applied  to  the  clinical 
diagnosis  of  syphilis  it  had  been  demonstrated  in  numerous 
other  infections  by  Bordet  and  Gengou.  The  test  is  applicable 
in  cholera,  typhoid,  whooping  cough,  and  other  diseases,  but 
is  not  constant  during  the  infection,  and  is  only  strongly 
positive  during  convalescence.  The  test  is  considerably  used 
in  the  diagnosis  of  gonorrhoeal  infection  in  America.  The 
antigen  employed  is  derived  from  a  mixture  of  several  strains 
of  gonococci.  In  human  tuberculosis  no  satisfactory  diagnostic 
test  of  this  nature  has  yet  been  published,  though  several 
methods  are  under  consideration.  The  difficulty  lies  in  the 
preparation  of  a  satisfactory  antigen. 

In  hydatid  disease  the  test  may  be  used  as  a  elinical  method 


80  CLINICAL   PATHOLOGY. 

of  diagnosis.  The  antigen  is  here  the  fluid  from  a  hydatid 
cyst,  and  if  this  be  substituted  for  the  lipoid  extract  the  test 
for  the  presence  of  hydatid  infection  may  be  carried  out  in  the 
same  manner  as  the  Wassermann  test  for  syphilitic  infection. 
On  theoretical  grounds  it  might  be  expected  that  the  com- 
plement fixation  test  would  be  applicable  in  all  diseases  in 
which  the  specific  infecting  body  or  antigen  is  known;  in 
practice  the  test  is  at  present  almost  entirely  confined,  with 
the  exception  of  hydatid  infections,  to  the  diagnosis  of  syphilis. 
Both  in  tuberculosis  and  in  gonorrhoea  the  test  is  still  under 
consideration. 

The  test  has  been  used  in  a  similar  manner  to  the  agglutina- 
tion test  as  the  specific  proof  of  an  infecting  organism.  The 
bacillus  isolated  by  Bordet  and  Gengou  from  cases  of  whooping 
cough  resembles  closely  the  influenza  bacillus,  but  by  means 
of  the  complement  fixation  test  they  have  been  able  to 
prove  the  specificity  of  their  bacillus.  The  serum  of  patients 
convalescent  from  whooping  cough  unites  with  the  Bordet- 
Gengou  bacillus  and  fixes  complement,  but  does  not  unite 
with  the  influenza  bacillus. 


CHAPTEE   VI. 

THE    PARASITOLOGY    OF    THE    BLOOD. 

The  cultivation  of  bacteria  from  the  blood. — In  health 
the  blood  as  obtained  from  the  peripheral  circulation  is  sterile, 
and  the  demonstration  of  organisms  in  it  during  life  is  of 
pathological  significance.  A  migration  of  organisms  from  the 
tissues,  and  particularly  from  the  intestinal  tract,  into  the 
circulation  frequently  takes  place  shortly  before  death. 

Blood  cultures  should  be  made  in  the  following  conditions  : — 

Infective  endocarditis  ("Progressive,"  "Ulcerative," 
"  Malignant "  endocarditis)  differs  from  the  simple  acute 
and  the  simple  chronic  endocarditis  in  several  particulars. 
On  clinical  grounds  the  infective  form  may  be  diagnosed  by 
the  prolonged  and  intermittent  pyrexia  often  associated  with 
rigors,  by  the  variability  of  the  murmurs,  and  by  the  casting 
off  of  emboli,  which  may  lodge  in  the  spleen,  kidneys,  lungs, 
or  the  larger  arteries  or  veins.  The  main  pathological  dis- 
tinctions of  this  disease  consist  in  the  presence  of  organisms 
in  the  blood  and  the  nature  of  the  cardiac  lesions,  which 
differ  from  those  of  simple  endocarditis  in  being  associated 
with  an  actual  loss  of  substance  or  ulceration  of  the  cardiac 
valves  or  walls.  The  complete  proof  of  an  infective  as  opposed 
to  a  simple  endocarditis  rests  during  life  upon  a  demonstration 
of  the  causative  organism  in  the  blood.  In  simple  chronic 
endocarditis  the  blood  is  sterile,  and  a  positive  blood  culture 
means  that  the  condition  has  become  infective.  Unfortunately 
in  a  considerable  percentage  of  cases  of  infective  endocarditis 
the  organisms  cannot  be  recovered  by  the  ordinary  methods, 
and  a  negative  blood  culture  is  very  little  evidence  against  the 
infectivity  of  the  process. 

The  most  common  variety  of  infective  endocarditis  is  that 
which  occurs  in  rheumatic  subjects.  After  two  or  more 
attacks  of  rheumatic  fever  have  crippled  the  cardiac  valves 
the  disease  may  change  into  the  ulcerative  variety.  The 
organisms  obtained  from  the  blood  in  these  cases  are  almost 

p.  6 


82  CLINICAL  PATHOLOGY. 

invariably  streptococci,  which  differ  in  many  respects  from 
the  ordinary  Streptococcus  pyogenes.  The  exact  role  of  these 
streptococci  is  at  present  not  certainly  known.  It  is  held  by 
some  that  rheumatic  fever  is  a  streptococcal  infection,  and 
that  if  the  resistance  of  the  individual  fails,  the  local  lesion 
may  progress  to  ulceration  and  the  organisms  invade  the 
blood-stream  in  overwhelming  numbers.  Others  believe  that 
rheumatic  fever  is  a  disease  of  unknown  aetiology,  and  that 
the  streptococci  found  in  the  blood  in  ulcerative  endocarditis 
are  secondary  invaders  which  have  attacked  the  crippled  valves 
and  produced  ulceration  in  them. 

.  Less  commonly  ulcerative  endocarditis  may  arise  quite 
independently  of  rheumatic  infection,  and  may  be  associated 
with  organisms  other  than  streptococci,  as  the  pneumococcus, 
or  more  rarely  the  gonococcus.  The  pneumococcus  may  be 
present  in  the  blood  in  association  with  a  typical  ulcera- 
tive endocarditis,  and  may  be  found  as  a  sequel  to  lobar 
pneumonia. 

Primary  lesions  other  than  cardiac. — Organisms  may 
get  into  the  blood  from  numerous  peripheral  lesions  and  give 
rise  to  a  condition  which  has  been  known  as  septicemia.  In 
such  cases  recent  vegetations  may  be  found  on  the  cardiac 
valves,  just  as  localised  areas  of  inflammation  accompanied  by 
collections  of  organisms  may  occur  in  other  parts  of  the  body. 
Actual  ulceration  of  the  valves  and  of  the  heart  wall  may  also 
occur.  This  class  of  infection  is  best  considered  apart  from 
the  cases  of  ulcerative  or  infective  endocarditis,  in  which  the 
cardiac  lesion  is  primary  and  predominant.  These  infections 
include  puerperal  septicaemias,  in  which  the  organisms  gain 
entrance  through  the  genital  tract  and  may  follow  numerous 
surgical  conditions,  such  as  whitlow,  urethritis,  or  suppuration 
in  the  middle  ear.  The  organism  most  frequently  obtained  from 
the  blood  is  a  streptococcus.  The  coccus  usually  grows  readily, 
can  be  cultivated  in  the  great  majority  of  the  cases,  and  has 
all  the  characters  of  the  ordinary  Streptococcus  pyogenes. 
Organisms  less  commonly  obtained  are  the  pneumococcus,  and 
rarely,  in  cases  of  general  blood  infection  following  a  urethritis 
or  cervicitis,  the  gonococcus. 

General  diseases. — In  certain  general  infections  the  causa- 
tive organism  can  be  obtained  from  the  blood  at  some  time 
during  the  course  of  the  disease.     Pneumonia,  Malta  fever, 


THE  PARASITOLOGY  OF  THE  BLOOD.    83 

and  typhoid  fever  are  among  the  fevers  which  have  been  com- 
paratively recently  recognised  as  general  diseases  associated 
with  characteristic  local  lesions. 
The  value  of  the  results  obtained  from  blood  cultures. 

— -In  infective  endocarditis  a  positive  blood  culture  is  definite 
diagnostic  evidence  of  the  progressive  nature  of  the  cardiac 
lesion  ;  it  is  also  very  strongly  suggestive  of  a  fatal  termination 
in  the  near  future.  A  single  negative  result  is  no  evidence 
against  the  presence  of  infective  endocarditis.  Vaccines  may 
be  prepared  from  the  organisms  obtained,  but  it  is  now  recog- 
nised by  most  observers  that  vaccine  treatment  of  ulcerative 
endocarditis  is  practically  worthless,  and  if  any  other  mode  of 
treatment  in  any  way  capable  of  arresting  the  disease  were 
available  vaccines  would  be  gladly  abandoned. 

In  local  lesions  associated  with  clinical  evidence  of  a  general 
infection  the  cultivation  of  organisms  from  the  blood  confirms 
the  diagnosis,  and  is  naturally  of  serious  import.  A  consider- 
able proportion,  however,  of  such  cases,  in  which  the  Strepto- 
coccus pyogenes  is  obtained  from  the  blood,  recover,  and  a 
vaccine  should  be  prepared  and  given  as  soon  as  possible,  since 
there  is  reason  to  believe  that  vaccines  not  only  do  good,  but,  in 
conjunction  with  surgical  treatment  of  the  local  lesion,  they 
may  be  the  essential  cause  of  recovery. 

In  typhoid  fever  the  cultivation  of  the  blood  in  the  first 
week  of  the  disease  is  of  the  greatest  value.  The  early 
diagnosis  of  typhoid  is  difficult  on  clinical  grounds  alone,  and 
the  agglutination  test  does  not  become  positive  before  the  end 
of  the  first  week.  The  bacillus  may  be  isolated  from  the 
blood  within  the  first  day  or  two  of  the  attack  in  almost  every 
case,  and  may  be  tested  either  by  cultural  methods  or  by  the 
action  of  a  known  typhoid  serum  upon  it. 

In  estimating  the  significance  of  organisms  obtained  from 
the  blood  it  must  be  realised  that  the  bacteria  present  in  the 
culture  tubes  do  not  necessarily  come  from  the  circulation. 
Skin  contaminations  are  not  extremely  infrequent  in  skilled 
hands.  They  are  almost  the  rule,  if  great  care  is  not  taken 
with  the  technique.  The  organisms  most  frequently  obtained 
in  contaminated  cultures  are  staphylococci,  particularly  S. 
alius,  diphtheroid  bacilli,  and  bacillus  subtilis. 

The  mode  of  performing  a  blood  culture. — The  materials 
required  are  a  sterile  syringe  and  needle,  culture  media,  a 

6—2 


84  CLINICAL   PATHOLOGY. 

sterile  solution  of  citrated  saline,  sterile  swabs  and  a  towel,  a 
bandage,  an  alcoholic  solution  of  iodine,  and  a  paint-brush. 

The  syringe  should  be  an  all-glass  instrument  capable  of 
holding  10  c.c.  The  needle  should  be  a  moderately  stout  one, 
and  must  have  a  sharp  point.  It  is  as  well  always  to  be  provided 
with  two  needles  and  to  test  the  permeability  of  both  before 
sterilising.  The  syringe  is  taken  to  pieces,  and  each  piece  is 
lightly  wrapped  in  cotton  wool  to  prevent  bumping.  It  is  then 
placed  in  a  flat,  round  glass  dish  provided  with  a  cover  and  of 
such  a  size  that  it  can  be  conveniently  transported  after 
sterilisation.  Dish  and  syringe  are  placed  in  the  steriliser  or  in 
an  ordinary  saucepan  filled  with  water,  and  boiled  for  at  least 
half  an  hour.  The  needles  wrapped  in  cotton  wool  should  be 
dropped  into  the  water  during  the  last  5  minutes  of  the 
boiling.  When  the  water  is  cool  fit  the  syringe  together,  place 
in  the  glass  dish  and  fit  the  cover  on,  taking  care  to  touch  only 
the  external  surfaces  of  the  syringe  with  the  hands,  which 
have  previously  been  washed  with  soap  and  water. 

The  culture  medium  requisite  for  most  purposes  consists 
of  ordinary  beef  broth.  At  least  4  tubes  should  be  made 
ready. 

The  sterile  citrated  salt  solution  consists  of  normal 
saline  with  0*9  per  cent,  of  sodium  citrate  added.  Only  a  few 
cubic  centimetres  are  required,  and  these  should  be  contained 
in  a  small,  shallow  flask. 

The  iodine  solution  is  the  same  as  that  used  before 
ordinary  surgical  operations  and  consists  of  a  2  per  cent, 
solution  of  iodine  in  rectified  spirits  of  wine. 

The  blood  is  obtained  as  follows  :— Before  commencing 
make  sure  that  everthing  is  within  easy  reach,  and  if  possible 
obtain  the  help  of  an  intelligent  assistant.  The  patient  should 
be  lying  in  bed  with  the  arm  selected  supinated  and  drawn 
well  away  from  the  side,  but  resting  on  the  bed  or  on  a  pillow, 
and  with  the  face  turned  towards  the  opposite  shoulder.  Tie  a 
bandage  tightly  round  the  arm  in  such  a  way  as  to  compress 
the  main  vessels  and  tell  the  patient  to  clench  his  fist. 
Choose  the  largest  vein  about  the  bend  of  the  elbow ;  this  is 
almost  invaribly  the  median-basilic.  The  vein  can  be  readily 
seen  and  felt  in  almost  all  subjects,  but  occasionally  in  young 
well-nourished  women  it  is  possible  only  to  feel  the  vein.  If 
the  arm  is  (edematous  the  vein  may  be  neither  seen  nor  felt. 


THE  PARASITOLOGY  OF  THE  BLOOD.    85 

and  in  such  circumstances  it  is  advisable  to  expose  it  as  for 
an  ordinary  venesection.  When  the  vein  has  been  rendered 
prominent,  place  a  piece  of  waterproof  sheeting  under  the 
arm  and  paint  a  considerable  area  of  the  skin  over  and  round 
the  vessel  with  the  iodine  solution.  Wash  the  hands  throughly 
and  surround  the  patient's  arm  with  a  sterile  towel.  Take 
the  syringe  with  the  needle  firmly  attached  and  draw  up  about 
1  c.c.  of  the  citrated  salt  solution.  The  object  of  this  solution 
is  to  prevent  the  blood  clotting  in  the  needle.  Pass  the  needle 
slowly  and  steadily  through  the  skin  into  the  vein,  holding 
the  syringe  with  the  needle  pointing  in  the  opposite  direction 
to  the  blood  flow  and  the  barrel  of  the  syringe  as  nearly 
parallel  as  possible  with  the  patient's  forearm.  Directly  the 
needle  enters  the  lumen  of  the  distended  vein  the  blood  flows 
into  the  citrate  solution  and  the  syringe  must  then  be  held 
quite  still.  Withdraw  10  c.c.  of  blood  and  remove  the  needle 
and  syringe.  Immediately  press  a  sterile  swab  upon  the 
puncture  mark  and  hold  it  there  until  the  bandage  has  been 
released.  Divide  the  blood  among  the  4  broth  tubes,  placing 
1  c.c.  in  the  first  tube,  2  c.c.  in  the  second,  3  c.c.  in  the  third, 
and  4  c.c.  in  the  fourth.  It  is  found  that  by  varying  the 
proportions  of  blood  and  medium  in  the  culture  tubes  a 
growth  of  the  organisms  is  more  certainly  obtained,  and  it  not 
infrequently  happens  that  the  growth  only  takes  place  in  the 
tube  containing  the  least  blood.  Before  leaving  the  patient 
bandage  a  piece  of  sterile  gauze  over  the  puncture  wound,  and 
let  it  be  removed  the  next  day. 

The  culture  medium  employed  is  commonly  broth,  but  this 
must  naturally  be  varied  with  the  nature  of  the  organism 
sought  for.  In  the  case  of  suspected  gonorrhoeal  infection  it 
is  advisable  to  use  blood  serum  slope  cultures.  A  useful 
medium  to  employ  for  the  isolation  of  typhoid  bacilli  is  sterile 
ox  bile,  which  inhibits  by  virtue  of  its  salts  the  growth  of 
organisms  other  than  members  of  the  typhoid-coli  group. 
The  media  should  be  incubated  at  37°  C.  for  24  hours,  when 
a  sub-culture  is  made  from  each  broth  tube  on  to  an  agar 
slope  and  the  tubes  are  again  incubated.  The  majority  of 
organisms  grow  in  from  one  to  two  days,  but  some  of  the 
streptococci  found  in  cases  of  infective  endocarditis  grow  very 
slowly,  and  all  cultures  should  be  kept  at  least  7  days 
before  they  are  finally  pronounced  to  be  sterile.     Even  if  no 


86  CLINICAL   PATHOLOGY. 

visible  growth  be  seen  in  the  broth  it  is  advisable  to  make  and 
examine  films  and  to  sub-culture  at  intervals  on  to  solid  media. 
The  organisms  present,  having  been  obtained  in  pure  culture, 
should  be  examined  as  to  their  nature  by  the  ordinary  methods 
(Chapter  XL). 

Other  bacteria  which  may  exceptionally  be  obtained  from  the 
blood  are  the  tubercle  bacillus,  influenza  bacillus,  the  bacillus 
coii,  and  the  anthrax  bacillus.  The  isolation  of  the  tubercle 
bacillus  is  too  uncertain  to  be  of  clinical  value.  The  anthrax 
bacilli  may  in  the  terminal  stage  of  anthrax  septicaemia  in  the 
human  subject  be  extremely  numerous,  and  may  be  actually 
demonstrated  in  film  preparations.  In  no  other  infection  is 
the  demonstration  of  bacteria  in  blood  films  within  the  range 
of  ordinary  probability,  and  it  should  not  be  attempted. 

The  parasitology  of  the  blood  in  tropical  diseases. — 
Persons  who  have  lived  in  tropical  or  sub-tropical  climates 
may  on  their  return  to  this  country  still  harbour  in  their  blood 
parasites  with  which  they  have  become  infected.  Some 
acquaintance  with  the  more  important  blood  parasites  is 
therefore  necessary  in  making  a  clinical  diagnosis.  The  follow- 
ing brief  description  should  be  supplemented  by  reference  to 
the  larger  text-books  on  tropical  medicine  : — 

Malaria. — Ague  is  now  practically  non-existent  in  England, 
although  it  was  until  comparatively  recently  endemic  in  the 
fen  districts,  and  members  of  the  anophelinse,  the  mosquitoes 
responsible  for  the  spread  of  the  disease,  are  still  to  be  found 
there. 

Those  who  have  lived  in  malarial  countries  on  their  return 
to  England  are  apt  to  look  upon  any  febrile  condition  as 
malarial,  and  it  indeed  appears  that  many  who  have  been 
infected  over  long  periods  are  subsequently  liable  to  consider- 
able rises  of  temperature  from  comparatively  trifling  causes. 
It  is  very  exceptional,  however,  to  demonstrate  the  parasite  in 
the  blood  after  more  than  a  year's  residence  in  this  country. 
Patients  recently  returned  from  infected  countries  may  harbour 
the  parasites  in  considerable  numbers.  In  all  suspected  cases 
it  is  advisable  to  withhold  quinine  until  the  blood  has  been 
examined  in  order  that  the  diagnosis  may  be  confirmed  and 
the  type  of  organism  determined. 

The  malarial  parasites  are  three  in  number :  the  quartan, 
the  tertian,  and  the  benign  tertian.     Owing  to  the  frequency 


PLATE   V. 


,  j^. 

:;v;v 

•■"." 

j 

PLATE   V. 


Benign  Tertian.  Quartan. 


Sub-tertian  "Rings."  Sub-tertian  "Crescents. 

Malarial  Parasites. 
(Leishman's  Stain.) 


THE    PAKASITOLOGY   OF   THE'  BLOOD.  87 

of  mixed  infections  the  temperature  chart  is  not  as  a  rule  a 
sufficient  guide  to  the  nature  of  the  organism,  which  must  be 
identified  by  means  of  the  microscope.  The  quartan  and  the 
benign  tertian  forms  sporulate  in  the  peripheral  circulation,  and 
are  the  two  parasites  most  easily  confused.  The  quartan  is 
feebly  amoeboid,  its  pigment  granules  are  coarse,  and  the 
parasite  commonly  fills  the  red  cell  without  distending  it. 
The  rosette  contains  8  to  10  segments.  The  benign  tertian  is 
the  parasite  most  commonly  met  with  :  it  is  actively  amoeboid 
and  contains  fine  pigment  granules ;  it  only  partially  fills 
the  red  cell,  which  is  almost  always  enlarged,  frequently  shows 
polychromatophilic  degeneration  and  commonly  contains 
numerous  chromophilic  granules  known  as  Schuffner's  dots. 
The  rosette  contains  15  to  26  segments.  The  fresh  blood 
should  always  be  examined  in  order  to  observe  the  activity  of 
the  parasite  and  the  dancing  movements  of  the  pigment 
granules.  It  is  advisable  to  use  a  j^-inch  objective,  and  if  the 
blood  is  examined  immediately  a  warm  stage  is  unnecessary. 
The  rosette  forms  are  not  commonly  met  with  in  this  country. 
The  great  majority  of  the  parasites  in  a  preparation  made 
with0Leishman's  stain  show  an  irregularly-shaped  blue  body 
containing  a  small  knot  of  purple- staining  chromatin  and 
black  pigment  granules.  The  malignant  tertian  parasite  is  a 
more  serious  infection,  responds  less  readily  to  quinine,  is 
frequently  associated  with  a  very  grave  anaemia,  and  may  be 
complicated  by  blackwater  fever.  This  parasite  sporulates  in 
the  internal  organs  and  the  forms  present  in  the  peripheral 
circulation  are  readily  distinguished,  since  the  commonest 
appearances  met  with  are  the  so-called  signet  rings  and 
crescents.  In  Leishman-stained  preparations  the  "  signets  " 
show  as  small  delicate  blue  rings  with  a  knot  of  purple  chro- 
matin at  one  spot  on  their  circumference.  The  crescents  are 
blue  crescentic  bodies  containing  a  central  cluster  of  black 
pigment  granules.  The  crescents  appear  at  first  sight  to  be 
lying  free  in  the  blood,  but  on  closer  inspection  a  narrow  rim 
of  the  cytoplasm  of  the  red  blood  corpuscle  can  be  made  out, 
often  bridging  the  concavity  of  the  crescent.  The  malignant 
tertian  parasites  are  commonly  very  scanty  and  require  a 
prolonged  search  before  they  can  be  demonstrated. 

The  best  method  of  looking  for  all  forms  of  malarial  parasites 
is  to  obtain  the  fresh  blood  and  to  make  films  in  the  ordinary 


88  CLINICAL   PATHOLOGY. 

way.  The  films  should  be  stained  by  Leishman's  stain.  A 
prolonged  search  may  be  necessary,  and  if  the  parasites  cannot 
be  found  by  the  usual  methods  it  is  advisable  to  make  the 
films  as  thick  as  possible,  and  when  dry  to  hsemolyse  them 
by  dipping  them  in  tap-water  until  no  more  haemoglobin 
comes  out.  They  should  then  be  stained  in  carbol  thionin 
for  3  minutes,  washed  in  water  and  blotted  dry. 

Trypanosomiasis. — Trypanosome  infections  in  man  are 
rarely  met  with  and  never  arise  in  this  country,  but  the 
organisms  are  so  widely  spread  and  so  fatal  to  man  and  animals 
that  a  very  short  account  of  them  may  be  given  here.  Try- 
panosomes  are  to  be  found  in  the  blood  of  a  large  variety  of 
animals,  and  in  many  of  them  appear  to  produce  no  ill  effect, 
while  in  others  they  cause  disease  and  often  a  heavy  mortality. 
In  man  trypanosomiasis  is  the  cause  of  sleeping  sickness,  a 
disease  which  is  incurable,  has  almost  depopulated  vast  areas 
of  country,  and  is  still  extending  into  districts  previously  free. 

The  more  important  trypanosomes  are  the  following  : — 

T.  lewisi,  the  rat  trypanosome. 

T.  evansi,  which  attacks  camels,  elephants,  etc.,  and  is  the 
cause  of  the  disease  known  in  India  as  "  Surra."  0 

T.  brucei,  which  attacks  horses  and  bovines  and  produces 
the  disease  called  "  Nagana  "  in  Africa.  This  parasite  is  found 
also  in  the  native  antelopes,  which  appear  to  be  immune  to 
its  poison  and  to  act  as  reservoirs  for  the  infection  of  the 
domestic  animals.  T.  brucei  is  spread  by  a  tsetse  fly,  Glossina 
morsitans. 

T.  gambiense  is  the  cause  of  sleeping  sickness  in  man,  and 
is  spread  by  another  biting  fly,  Glossina  patyalis.  In  the 
early  stages  of  the  disease  the  trypanosomes  are  present  in  the 
blood  and  more  numerously  in  the  lymphatic  glands  and 
may  then  cause  few  symptoms.  It  is  only  in  the  late  stages 
that  the  organisms  gain  access  to  the  central  nervous 
system,  are  found  in  the  cerebro -spinal  fluid,  and  produce  the 
symptoms  of  the  disease.  It  may  be  mentioned  that  the 
most  constant  early  symptom  of  sleeping  sickness  is  insomnia. 

In  order  to  demonstrate  the  presence  of  the  trypanosomes 
in  a  suspected  case  the  blood  should  be  examined  both  in 
the  fresh  state  and  in  ordinary  films  stained  by  Leishman's 
stain.  The  parasite  is,  however,  frequently  scanty  in  the  peri- 
pheral  circulation,  and    it   may  be    necessary   to    withdraw 


THE  PAKASITOLOGY  OF  THE  BLOOD.    89 

several  cubic  centimetres  of  the  blood  from  a  vein,  mix  them 
in  citrated  salt  solution,  centrifuge,  and  examine  the  deposit. 
Usually  the  lymphatic  glands  are  enlarged,  and  most  frequently 
the  cervical  glands,  and  the  simplest  method  is  to  puncture  the 
most  prominent  gland  with  a  hypodermic  needle  and  syringe 
and  make  films  from  the  small  quantity  of  fluid  obtainable. 
By  this  method  trypanosomes  can  usually  be  demonstrated 
with  ease.  In  the  later  stages  the  cerebrospinal  fluid  may 
be  removed  by  lumbar  puncture,  centrifuged,  and  the  deposit 
examined.  An  excess  of  lymphocytes  is  present  in  the  fluid 
in  addition  to  the  parasites  themselves.  If  parasites  cannot 
be  found  by  any  of  these  methods,  the  conclusive  proof  of 
absence  of  infection  rests  upon  the  inoculation  of  susceptible 
animals  with  the  patient's  blood. 

The  trypanosome  as  seen  in  the  fresh  blood  is  actively 
amoeboid  and  provided  with  a  free  flagellum  at  its  posterior 
extremity.  In  stained  preparations  the  following  points  may 
be  made  out.  Near  the  anterior  rounded  extremity  is  a 
small,  deeply  staining  round  spot,  the  blepharoblast.  Posterior 
to  the  blepharoblast  is  a  vacuole,  and  posterior  to  this  again 
is  the  nucleus,  which  is  commonly  situated  near  the  centre 
of  the  trypanosome.  The  undulating  membrane  can  be  seen 
arising  from  the  blepharoblast,  winding  along  the  free  border 
of  the  parasite  and  terminating  in  the  flagellum  at  the 
posterior  extremity.  The  identification  of  the  different  species 
of  trypanosomes  must  be  left  to  the  expert. 

Leishmania. — Leishmania  is  in  reality  a  variety  of  try- 
panosomiasis. The  mature  trypanosome,  however,  is  in  this 
instance  absent  from  the  human  tissues  and  is  represented  by 
a  developmental  form  known  as  the  Leishman-Donovan  body. 
The  disease  produced  is  known  as  kala-azar  or  black  fever, 
is  almost  invariably  fatal,  and  is  accompanied  by  considerable 
pyrexia  and  marked  enlargement  of  the  spleen.  The  diag- 
nosis of  the  disease  from  malaria  and  other  causes  of  splenic 
enlargement  rests  upon  the  demonstration  of  the  parasites.  The 
organisms  may  be  searched  for  in  the  blood,  in  which  they 
may  be  found  within  the  leucocytes  and  usually  the  large 
hyaline  cells.  The  parasites,  however,  are  commonly  scanty 
in  the  peripheral  circulation,  and  it  may  not  be  possible  to 
find  them  even  after  a  prolonged  search.  The  most  practical 
method  is  to  perform  puncture  of    the  spleen,  a  proceeding 


90  CLINICAL   PATHOLOGY. 

which  has  in  the  past  been  attended  by  fatal  results  owing 
to  the  wounding  of  considerable  blood  vessels  with  a  large 
needle.  If  puncture  of  the  spleen  is  performed  with  the 
ordinary  hypodermic  syringe  armed  with  the  usual  fine  needle, 
the  operation  is  practically  devoid  of  risk  and  the  minute 
quantity  of  splenic  fluid  obtained  is  quite  sufficient  for  diag- 
nostic purposes.  Films  should  be  made  from  the  fluid  and 
stained  with  Leishman's  stain.  The  organism  is  found  for 
the  most  part  within  the  splenic  cells  and  appears  as  a  small, 
rounded  body  containing  a  round  nucleus  and  a  small,  deeply 
staining  rod-shaped  micronucleus  or  blepharoblast.  The 
organism  can  be  cultivated  outside  the  body  in  citrated  blood 
at  20°  C,  and  flagellated  trypanosomes  are  produced  from  the 
Leishman-Donovan  bodies.  It  is  probable  that  the  organism 
usually  passes  through  its  developmental  stage  in  a  bug,  and 
is  by  this  agency  inoculated  from  one  human  being  to  another. 
Similar  bodies  are  present  in  cases  of  "  Oriental  sore  "  and  in 
other  varieties  of  European  splenomegaly. 

Spirochetosis — The  only  spirochaBte  which  can  be  demon- 
strated in  man  in  the  circulating  blood  is  that  of  relapsing 
fever,  known  as  the  spirochceta  recurrentis  or  the  spirillum 
Obermeieri.  There  is  every  reason,  however,  to  suppose  that  the 
European  form,  the  spirochceta  recurrentis,  differs  from  the 
Egyptian,  the  African,  the  American  and  the  Asiatic  varieties. 
The  disease  is  accompanied  by  febrile  attacks  of  5  to  7  t days' 
duration,  followed  by  periods  of  apyrexia  and  one  to  two  relapses. 
The  spleen  is  usually  enlarged.  Eelapsing  fever  is  still  common 
in  Russia  and  other  parts  of  Europe,  but  has  become  practically 
non-existent  in  this  country,  although  cases  are  still  to  be  met 
with  in  Ireland.  The  mortality  rate  is  under  15  per  cent. 
The  spirochaBte  is  considerably  coarser  than  the  syphilitic 
organism,  and  the  spirals  are  less  regular ;  it  has  a  length  of 
about  12  fi  and  is  actively  amoeboid.  The  fresh  blood  during 
the  febrile  stages  shows  the  organisms  in  large  numbers  ;  they 
are  also  readily  recognised  in  stained  films. 

Filariasis. — The  most  common  embryo  of  the  nematode 
worms  which  may  be  found  in  the  blood  of  man  is  that  of 
F.  iioctuma.  The  parent  worms  live  in  the  lymphatics  of  the 
limbs  or  trunk,  and  pass  their  young  into  the  lymphatic  stream 
and  so  into  the  blood.  The  parent  worms  by  blocking  the 
lymphatic  circulation  may  give  rise  to  elephantiasis,  or,  in  the 


PLATE   VI. 


r       v    m  Tr/panosomes-  Leishman-Donovan  Bodies. 

Camel  s  Blood.     (Lehman's  Stain.)  (Leishman's  Stain.) 


Spirilla  of  Eelapsmg  Fever.  Filaria  and  sheath 

CJ^ZS     t       ^  HEemolysed  Blood  Film. 

(LeishmansStam.)  (Hematoxylin.). 


PLATK    VI. 


THE  PARASITOLOGY  OF  THE  BLOOD.    91 

rare  cases  in  which  they  are  located  in  the  bladder,  to 
chyluria.  In  the  majority  of  cases,  however,  they  give  rise  to  no 
symptoms.  The  embryos  appear  in  large  numbers  in  the 
peripheral  blood  at  night,  and  during  the  daytime  retire  to 
the  heart  and  large  vessels.  The  embryos  in  the  blood  are 
enclosed  in  a  sheath  from  which  they  cannot  escape,  but  within 
which  they  can  move.  The  embryos  are  sucked  from  the 
blood  by  the  mosquito  (the  culex  fatigans),  and  in  its  stomach 
get  rid  of  their  sheath.  They  subsequently  bore  their  way 
through  the  stomach  wall  of  the  mosquito  and  pass  into  the 
thoracic  muscles,  where  they  undergo  further  development. 
The  worm  then  works  its  way  into  the  proboscis  of  the  mosquito, 
and  so  passes  again  to  man.  The  filariae  of  man  are 
represented  by  three  main  species,  F.  nocturna,  F.  diurna  and 
F.  pcrstans.  The  embryos  should  be  looked  for  in  the  fresh 
blood  and  in  stained  preparations  made  from  comparatively 
thick  films.  In  the  case  of  F.  nocturna  the  blood  should  be 
examined  between  7  o'clock  at  night  and  7  o'clock  in  the 
morning.  The  embryos  are  easily  recognised,  and  should  be 
sought  for  under  a  low  power  (e.g.,  f-inch  objective)  of  the 
microscope.  For  the  differentiation  of  the  various  species  the 
student  should  refer  to  works  on  tropical  medicine. 


CHAPTER   VII. 

THE    CHEMICAL    AND    PHYSICAL    EXAMINATION    OF    THE    BLOOD. 

The   spectroscopic   examination   of   the  blood. — In 

order  to  examine  the  blood  in  this  way  all  that  is  necessary 
is  to  prick  the  patient's  thumb  and  squeeze  3  or  4  drops 
of  blood  into  a  clean  beaker  containing  about  10  c.c.  of  distilled 
water.  A  portion  of  the  mixture  is  then  transferred  to  a  test 
tube  and  diluted  until  it  is  of  a  pale  pink  colour.  The  test 
tube  is  then  examined  with  a  direct  vision  spectroscope. 

The  normal  spectrum  of  the  blood  is  of  course  that  of 
oxyhemoglobin,  and  the  two  characteristic  absorption  bands 
are  placed  between  the  D  and  E  lines.  The  band  nearest  the 
D  line  is  darker,  narrower,  and  more  sharply  defined  than  the 
other  band.  On  adding  a  few  drops  of  ammonium  sulphide 
to  the  test  tube,  inverting  the  tube  several  times  and  allowing 
it  to  stand  for  a  few  minutes,  the  two  absorption  bands  become 
merged  into  the  one  band  of  reduced  hemoglobin. 

Carbonic  oxide  poisoning  may  occur  after  exposure  to 
coal  gas  or  to  the  fumes  of  charcoal  stoves.  The  colour  of 
the  blood  in  these  cases  is  brighter  than  normal,  and  has  a 
characteristic  cherry-red  appearance.  The  diagnosis  of  the 
condition  is  made  certain  by  an  examination  of  the  spectrum. 
The  absorption  bands  of  carboxyhsemoglobin  are  two  in  number, 
and  are  situated  slightly  nearer  the  violet  end  of  the  spectrum 
than  those  of  oxyhemoglobin.  The  difference  in  position  of 
the  carboxyhernoglobin  and  the  oxyhemoglobin  bands  is,  how- 
ever, so  slight  as  to  be  appreciated  with  difficulty  when  working 
with  a  small  spectrum.  The  nature  of  the  spectrum  is  definitely 
determined  by  adding  ammonium  sulphide  and  finding  that  no 
alteration  takes  place  in  the  bands,  which  remain  distinct  and 
separate  since  the  carbonic  oxide  combination  with  hemoglobin 
is  more  stable  than  that  of  oxygen. 

Methaemoglobinsemia  may  be  produced  by  certain  drugs, 
particularly  phenacetin  and  antipyrin,  even  when  taken  in 
medicinal  quantities  by  susceptible  patients.  The  same 
condition   may   arise   among    workers    in   aniline   dyes   and 


PLATE   VIL 


C                        D                         -E   T) 

\65                     60    1                  55 

50 

^H 

JS                     60 

II    III    1 

^^^ 

50 

//'■ 

65                     60 

|  1    |    I  |  |     1     |     1     |      |     | 

^ 

50 

fif                     60                  |    55 

50 

1  IT  I    I    I    I    i    ill    1    1    i    I       |  i  i                  iii 

'  1 

Iii  Jgfi     liTiinfi  ii    i    f 

1 

1      ■! 

|  j  i   I  |  I    ;    1    |    |     Mill     1,1,! 

^^^^R     ■          ^H     KSfl^H 

..      65                     SO         ,     ,    ' ,    55    .      , 

■50        , 

!///■ 

65                     60                       55                                50 

^^^^^^^■flsoMty^x-yi 

■  ki  ,  i  i  f  i  i      f  i 

5p 

xl 

1 

Oxyhemoglobin. 


Hemoglobin. 


Carboxyhremoglobin. 


Methaemoglobin. 


HaBmochromogen  in  alkaline 
solution. 


Hematin  in  acid  alcohol. 


Acid  HsBmatoporphyrin. 


Alkaline  Hemalxmorphyrin. 


Urobilin. 


Uroerythrin. 


D 


E    h 


Absorption  Spectra. 


(From  Hoppe-Seyler-Thierfelder's  "  Handbuch  der  Physiologisch-und- 
Pathologisch-Chernischen  Analyse.") 


EXAMINATION   OF   THE   BLOOD.  93 

nitro-glycerin  factories  from  the  inhalation  of  nitrobenzol  com- 
pounds, and  may  follow  poisoning  by  chlorate  of  potash  and 
by  pyrogallic  acid.  The  patients  are  markedly  cyanosed,  and 
the  colour  of  the  blood  in  severe  cases  is  distinctly  brownish. 
It  may  be  added  that  in  a  considerable  proportion  of  cases  of 
hemoglobinuria  the  hemoglobin  is  present  in  the  urine  as 
methemoglobin.  Methemoglobin  contains  the  same  amount 
of  oxygen  as  oxyhemoglobin,  but  in  different  combination ;  it 
can  be  produced  artificially  by  adding  a  few  drops  of  potassium 
ferricyanide  to  diluted  blood  and  warming  gently.  It  is  well, 
in  cases  of  doubt,  to  make  an  artificial  solution  of  this  nature 
for  purposes  of  comparison.  The  characteristic  absorption 
band  of  the  spectrum  is  a  narrow,  sharply  denned  band  in  the 
red  (between  C  and  D).  In  dilute  solution  other  bands  apjiear, 
including  two  bands  corresponding  to  those  of  oxyhemoglobin. 
On  adding  ammonium  sulphide  to  the  methemoglobin  solu- 
tion, the  band  in  the  red  disappears  at  once,  and  the  two  bands 
of  oxyhemoglobin  more  slowly  merge  into  the  single  band  of 
hemoglobin. 

Sulph-haemoglobinsemia  is  an  extremely  rare  condition, 
accompanied  clinically  by  considerable  cyanosis.  There  are 
very  few  recorded  cases,  and  there  is  reason  to  believe  that 
all  of  these  are  not  genuine.  Sulph-hemoglobin  can  be 
artificially  produced  by  the  addition  of  a  small  volume  of 
sulphuretted  hydrogen,  and  it  has  been  suggested  that  the 
origin  of  the  condition  should  be  sought  in  the  intestinal 
tract.  The  spectrum  of  sulph-hemoglobin  gives  a  band  in 
the  red  similar  to  that  of  methemoglobin,  but  nearer  the 
violet,  as  well  as  the  two  bands  of  oxyhemoglobin.  On  adding 
ammonium  sulphide  the  band  in  the  red  persists,  while  the 
two  bands  of  oxyhemoglobin  slowly  merge  into  one. 

The  Chemical  Examination  of  the  Blood. 

Lipaemia. — By  lipemia  is  meant  the  presence  of  fat  in 
readily  demonstrable  amount  in  the  blood.  The  condition  is 
a  rare  one,  and  has  been  described  in  a  variety  of  affections 
including  tuberculosis,  alcoholism,  and  nephritis.  The  disease 
most  frequently  associated  with  lipemia,  however,  is  diabetes, 
and  particularly  that  variety  of  diabetes  which  attacks  young 
subjects.  The  condition  has  been  diagnosed  clinically  by 
direct  observation  of  the  fat  droplets  circulating  in  the  retinal 


94  CLINICAL  PATHOLOGY. 

vessels.  Lipsemia  can  be  recognised  by  withdrawing  blood 
in  a  serum  tube  in  the  same  manner  as  for  a  Widal  reaction 
and,  after  allowing  to  stand  for  half  an  hour,  centrifuging  at 
a  moderate  speed.  The  blood  obtained  in  this  way  has  a  most 
characteristic  appearance,  the  serum  loaded  with  fat  forming 
an  opaque,  turbid  layer  above  the  red  cells.  It  is  necessary 
to  prove  that  the  turbidity  of  the  serum  is  due  to  fat.  A  few 
drops  of  the  serum  should  be  placed  on  a  slide  with  a  cover- 
slip  over  them  and  examined  with  the  microscope  for  the 
presence  of  fat  droplets.  In  addition,  films  should  be  made 
on  slides,  fixed  in  formalin  vapour  for  15  minutes,  stained 
with  Scharlach  E.  for  from  12  to  21  hours,  dipped  in  75  per  cent, 
spirit  for  a  few  seconds,  washed  in  distilled  water  and  mounted 
in  Farrant's  medium.    The  fat  droplets  are  stained  a  bright  red. 

In  certain  other  conditions,  and  particularly  in  chronic 
nephritis  with  cedema,  a  serum  may  be  obtained  from  the 
blood  milky  in  appearance  and  strongly  opalescent.  The 
serum  separates  rapidly  and  may  be  mistaken  for  a  fatty 
serum.  The  opalescence  in  these  cases  is  not  due  to  free  fat, 
but  to  an  unknown  substance  of  a  proteid  nature.  A  similar 
opalescence  may  be  present  in  other  body  fluids,  and  when 
found  in  fluid  withdrawn  from  the  peritoneal  cavity  has  to 
be  distinguished  from  the  veiy  much  rarer  condition  of 
chylous  or  fatty  ascites. 

Glycogensemia  (iodophilia). — The  iodine  reaction  in 
the  blood  consists  in  the  presence  of  iodophil  granules  within 
the  polynuclear  neutrophil  leucoc}Ttes.  There  has  been 
considerable  dispute  as  to  the  nature  of  these  granules,  but 
the  consensus  of  opinion  would  seem  to  be  that  they  are 
composed  of  glycogen  either  in  combination  or  lying  free 
within  the  cells.  A  positive  iodophil  reaction  may  occur 
under  a  variety  of  conditions,  but  is  very  constantly  present 
in  all  tox&emias,  and  particularly  in  septic  infections  accom- 
panied by  suppuration.  The  reaction  has  been  largely  used 
as  a  clinical  method  of  diagnosing  the  presence  of  pus,  but 
owing  to  the  wide  varietj^  of  causes  capable  of  producing  the 
condition,  as  well  as  to  the  varying  degrees  of  intensity  of  the 
reaction  itself  and  the  consequent  difficulty  of  interpreting 
the  results,  a  positive  reaction  is  of  no  great  assistance.  In 
cases  of  doubtful  suppuration  a  negative  iodophil  reaction  is 
strongly  opposed  to  the  presence  of  pus. 


EXAMINATION   OF   THE   BLOOD.  95 

The  reaction  is  performed  as  follows  :  a  blood  film  is  made 
in  the  ordinary  way  on  a  cover- slip  and  dried  in  the  air  ;  the 
film  is  then  mounted  in  and  simultaneously  stained  by  the 
following  solution  : — 

Iodine  .         .         .         .  .       .         .1  gramme 

Potassium  iodide .         .         .         .         .3  grammes 

Distilled  water 100  c.c. 

Gum  acacia,  sufficient  to  make  a  mixture  of  about 
the  consistence  of  Farrant's  solution. 

The  smallest  possible  quantity  of  the  fluid  as  a  mountant 
should  be  used  in  order  to  avoid  opacity. 

Preparations  made  in  this  way  will  keep  for  several  weeks. 

On  examining  the  preparations  with  an  oil  immersion  lens 
two  kinds  of  reaction  are  seen,  an  extra-cellular  and  an  intra- 
cellular. The  extra-cellular  reaction  consists  of  purple-brown 
amorphous  fragments  lying  free  in  the  plasma,  and  is  of  little 
significance  since  it  is  present  in  normal  blood.  The  intra- 
cellular reaction  is  practically  confined  to  the  polynuclear 
neutrophils  and  varies  from  a  diffuse  brown  staining  of  the 
cytoplasm  due  to  the  presence  of  innumerable  minute 
iodophil  granules,  to  the  appearance  of  coarse,  intensely 
stained,  purple-brown  granules  in  the  periphery  of  the  cells. 
The  polynuclears  of  normal  blood  stained  by  iodine  show  only 
a  faint  lemon-yellow  colour  in  the  cytoplasm.  The  red  cells 
stain  a  faint  orange  and  show  no  variation  in  normal  or  in 
pathological  blood. 

Cholsemia. — By  cholremia,  in  this  connection,  is  simply 
meant  the  presence  of  bile  constituents  in  the  blood.  In 
most  conditions  associated  with  jaundice  the  amount  of  bile 
pigment  in  the  blood  is  commonly  sufficient  to  colour  the 
skin  and  conjunctivae  to  such  a  degree  as  to  render  any  special 
examination  unnecessary.  The  pigment,  however,  can  be 
demonstrated  in  the  serum  long  before  definite  jaundice  can 
be  recognised  on  clinical  grounds.  The  amount  of  bile  which 
may  be  present  in  the  blood  in  some  cases  of  cirrhosis  of  the 
liver,  pernicious  anaemia  and  congenital  family  cholaBmia  can 
commonly  only  be  detected  by  a  chemical  examination  of  the 
blood.  Such  small  amounts  of  pigment  are  also  rarely 
met  with  in  apparently  normal  individuals  as  a  periodic 
phenomenon. 

To   test   for   the   presence   of   bile  pigment   in  the  blood 


96  CLINICAL   PATHOLOGY. 

withdraw  a  sample  of  blood  in  a  serum  tube,  stand  for  a  time 
and  centrifuge.  The  presence  of  bile  is  rendered  quite 
evident  by  the  bright  yellow  colour  of  the  supernatant  serum. 
The  colour  of  the  serum  is  hardly  recognisable  by  artificial 
light  and  should  always  be  examined  by  daylight,  a  precaution 
which  applies  equally  to  the  clinical  examination  of  a 
jaundiced  person.  The  coloration  of  the  serum  by  bile 
pigment  can  be  confounded  with  that  of  a  serum  tinged  with 
haemoglobin,  as  may  occur  if  the  blood  is  carelessly  withdrawn 
and  centrifuged.  The  bile  colour  is  more  closely  simulated 
by  the  greenish-yellow  pigment  present  in  the  serum  of 
nearly  all  cases  of  pernicious  anaemia.  A  certain  percentage 
of  these  cases  are  in  addition  actually  jaundiced.  The 
presence  of  bile  pigment  should  always  be  confirmed  by  a 
chemical  test  such  as  Gmelin's.  The  serum  is  pipetted  off 
and  allowed  to  soak  into  as  concentrated  an  area  as  possible 
of  a  clean  filter  paper ;  a  drop  of  fuming  nitric  acid  is  then 
placed  in  the  centre  of  the  serum  area  and  the  play  of  colours 
— green,  blue,  red  and  yellow — looked  for  in  the  rings  which 
form  at  the  junction  of  acid  and  serum. 

Uric  acid  in  the  serum. — The  presence  of  uric  acid  in 
the  blood,  in  excess,  can  be  demonstrated  by  the  following 
time-honoured  experiment.  A  few  cubic  centimetres  of  serum 
are  placed  in  a  watch-glass  and  made  distinctly  acid  with 
28  per  cent,  acetic  acid.  One  or  two  threads  of  cotton  are 
left  soaking  in  the  mixture,  which  is  allowed  to  evaporate  at 
room  temperature.  Crystals  of  uric  acid  deposit  on  the 
threads  in  about  24  hours.  The  reaction  is  most  commonly 
successful  in  cases  of  gout,  but  is  of  no  particular  clinical 
significance,  since  it  may  fail  to  appear  in  the  blood  of 
obviously  gouty  patients  and  may  be  present  in  a  considerable 
variety  of  other  conditions. 

The  exact  estimation  of  the  uric  acid  in  the  blood  has  no 
serious  application  in  medicine. 

The  specific  gravity  of  the  blood. — The  specific 
gravity  of  the  normal  blood  is  about  1*060,  and  except  in  cases 
of  dropsy  varies  directly  with  the  percentage  of  the  haemo- 
globin. The  estimation  of  the  specific  gravity  is  of  very  little 
clinical  importance,  but  has  been  made  use  of  in  the  past 
largely  as  a  method  of  estimating  the  haemoglobin.  The 
simplest  method  of  taking  the  specific  gravity  is  to  prepare  a 


EXAMINATION   OF   THE    BLOOD.  97 

mixture  of  chloroform  and  benzol  of  a  specific  gravity  of  l-060, 
and  add  a  drop  of  blood  to  the  mixture.  If  the  blood  (which 
does  not  mix  with  these  liquids)  sinks  to  the  bottom,  add 
chloroform  ;  if  the  drop  rises  to  the  top,  add  benzol  until  a 
mixture  is  obtained  in  which  the  drop  of  blood  remains 
stationary  in  the  body  of  the  liquid.  The  specific  gravity  of 
this  mixture  is  the  specific  gravity  of  the  blood. 

The  alkalinity  of  the  blood. — The  degree  of  alkalinity 
of  the  blood  is  fairly  constant  in  health  and  varies  considerably 
in  an  important  group  of  morbid  conditions  which  includes 
diabetic  coma  and  the  toxaemias  of  pregnancy.  The  estima- 
tion of  the  alkalinity  of  the  blood  has  not,  however,  come  into 
general  clinical  use,  partly  because  no  simple  and  at  the  same 
time  accurate  method  has  yet  been  devised.  For  this  pur- 
pose Wright's  method  or  one  of  its  modifications  may  be 
used,  but  it  must  be  acknowledged  that  little  information  of 
any  clinical  value  is  to  be  obtained  from  such  methods. 

The  following  technique  may  be  adopted. 

Obtain  a  sample  of  blood  serum  in  the  ordinary  way.   Have 

ready  a  series  of  small  bottles  containing  standard  solutions  of 

N  N   N  N 

sulphuric  acid  ranging  from  -^-^-^     .     .     .     — .      To  each 

o    I'D    1  o 

bottle  of  acid  add  1  or  2  drops  of  dimethyiamidoazobenzol. 

In  a  Wright's  pipette  take  up   1  volume  of  the  serum  and 

1  volume  of  one   of    the    acid    solutions.      Mix  thoroughly 

together  in  a  white  porcelain  dish.     If  the  resulting  mixture 

is   yellow   repeat   with   a   stronger  acid   until   a   mixture   is 

obtained  with  a  distinctly  orange-red  tinge.     That  strength  of 

acid  is  taken  as  the  equivalent  of  the  alkalinity  of  the  serum 

which  last  gave  the  yellow  colour.      The  index  of  alkalinity  is 

recorded  as  the  fraction  of  the  equivalent  acid  solution.     For 

.    N 
example,  if  —  acid  neutralises  the  serum  the  index  is  OI66.    In 

normal  cases  the  average  is  0*170. 

In  estimating  the  alkalinity  by  this  method  the  blood  should 
be  withdrawn  3  hours  after  a  meal,  and  the  serum  tubes  and 
pipettes  used  should  have  been  soaked  in  hydrochloric  acid 
to  remove  all  alkali,  repeatedly  washed  in  distilled  water  and 
finally  dried  in  an  oven  at  120°  C. 

The  estimation  is  rapidly  performed,  but  the  recognition  of 
the  neutral  point  in  the  reaction  is  difficult  when  dealing  with 

p.  7 


98  CLINICAL   PATHOLOGY. 

such  small  quantities,  and  the  experimental  error  is  consider- 
able. 

The  oxygen  content  of  the  blood. — The  estimation  of 
the  oxygen  content  of  the  blood,  and  from  it  the  total  volume 
of  the  blood  in  the  body,  has  been  rendered  possible  by  the 
researches  of  Haldane  and  Lorraine  Smith.  The  method 
which  they  employ  is  beyond  the  scope  of  ordinary  clinical 
pathology,  and,  moreover,  depends  upon  an  inhalation  by  the 
patient  of  a  volume  of  carbonic  oxide  gas,  a  proceeding  which 
is  certainly  not  devoid  of  risk  in  unaccustomed  hands.  The 
method  may  be  briefly  indicated  here.  The  patient  is  made 
to  inhale  a  measured  volume  of  carbon  monoxide  and  a  few 
minutes  later  a  sample  of  his  blood  is  taken  and  the  extent 
to  which  the  haemoglobin  has  become  saturated  with  the  CO 
is  estimated.  From  the  amount  of  gas  inhaled  and  the  extent 
to  which  the  haemoglobin  has  become  saturated  in  the  sample 
the  quantity  of  CO  capable  of  being  taken  up  by  the  whole  of 
the  patient's  blood  can  be  calculated.  For  example,  if  100  c.c. 
of  CO  were  inhaled  and  the  sample  is  one-fifth  saturated,  the 
blood  would  have  been  100  per  cent,  saturated  by  500  c.c.  and 
the  capacity  of  the  blood  for  CO,  and  similarly  the  oxygen 
capacity  of  the  blood  would  be  500  c.c. 

The  estimation  of  the  total  volume  of  blood  in  the  body  can 

be  farther  determined  by  comparing  the  colour  of  the  patient's 

blood  with  the  colour  of  an  equal  sample  of  ox  blood,  the 

oxygen  capacity  of  which  has  been  previously  determined.     If, 

for  example,  the   patient's  blood   has  the  same   colour  as  a 

sample  of  ox  blood  every  100  c.c.  of  which  has  been  found 

capable  of  absorbing  20  c.c.  of  oxygen,  and  the  total  oxygen 

capacity  of  the  patient's  blood  is  500  c.c,  since  20  c.c.  of  oxygen 

can  be  taken  up  by  100  c.c.  of  his  blood  and  his  total  blood 

can   take   up   500   c.c.  of  oxygen,  his   total   blood   measures 

100  X  500       0  KAA 
^r or  2,500  c.c. 

The  total  volume  of  blood  in  health  has  been  found  to  vary 
between  3,000  and  4,500  grammes,  or  from  one-thirtieth  to 
one-sixteenth  of  the  body  weight. 

In  chlorosis  the  total  oxj-gen  capacity  of  the  haemoglobin 
remains  normal,  while  the  total  volume  of  the  plasma  is 
greatly  increased ;  consequently  the  percentage  of  haemoglobin 
as  estimated  in  the  usual  way  is  low,  but  is  actually  only 


EXAMINATION   OF    THE   BLOOD.  99 

relatively  diminished  in  proportion  to  the  bulk  of  blood. 
In  pernicious  anaemia,  on  the  contrary,  the  haemoglobin  is 
actually  diminished.  The  most  marked  increase  in  the  total 
volume  of  blood  occurs  in  Osier's  disease,  or  splenic  poly- 
cythsernia,  in  which  the  blood  volume  may  reach  three  times 
that  of  the  normal.  In  this  disease,  as  already  mentioned,  the 
numbers  of  red  cells  to  the  cubic  millimetre  may  be  double  the 
normal,  so  that  the  actual  number  of  red  cells  in  the  whole 
body  may  be  6  times  the  normal  amount. 

It  has  been  indicated  that  no  purely  chemical  examination 
of  the  blood  has  at  the  present  any  wide  application  in 
clinical  medicine.  Certain  methods,  however,  which  have 
been  described  in  previous  chapters  might  properly  have  been 
included  here.  A  differential  leucocyte  count,  for  example, 
depends  upon  the  chemical  affinity  of  the  cell  and  its  granules 
for  different  dyes.  Such  a  widely  used  test  as  the  Wassermann 
reaction  might  also  be  considered  as  a  chemical  process.  It  is 
further  probable  that  other  and  more  strictly  chemical  methods 
of  examination  will  come  into  general  use  in  the  near  future. 
No  description  has  been  given  here  of  the  estimation  of  the 
various  ferments  in  the  blood,  but  a  considerable  amount  of 
experimental  work  in  this  direction  has  recently  been  per- 
formed, and  the  relation  between  carcinoma  and  the  amount 
of  trypsin  in  the  blood  has  been  made  use  of  as  a  method  of 
diagnosis.  The  variations  in  the  amount  of  this  ferment  in 
carcinoma  are,  however,  small,  and  since  the  causes  of  such 
variations  are  by  no  means  confined  to  carcinoma,  it  may  be 
said  that  the  method  has  little  clinical  value  at  the  present. 


7—2 


SECTION    II. 

BACTERIOLOGY. 

CHAPTEE  YIII. 

Introductory — Table  of  Classification. 

CHAPTEE  IX. 

The  Cocci — The  Gram -positive  Bacilli. 

CHAPTEE  X. 
The  Grani-negative  Bacilli — Spirilla — Streptothricese — Hyphomycetes. 

CHAPTEE  XI. 

Bacteriological  Methods— General  and  Special. 

CHAPTEE  XII. 

Vaccines — An  ti- sera. 

CHAPTEE  XIH. 

Preparation  of  Culture  Media — Staining  Eeagents. 


CHAPTER  VIII. 

INTRODUCTORY — TABLE    OF    CLASSIFICATION. 

Before  considering  the  application  of  bacteriology  and 
bacteriological  methods  in  the  diagnosis  and  treatment  of 
disease  it  is  necessary  to  describe  as  briefly  as  possible  the 
general  means  at  our  disposal  for  the  investigation  of  the  chief 
pathogenic  bacteria.  For  the  identification  of  bacteria  we 
are  dependent  upon  a  study  of  their  morphology,  staining 
properties  and  cultural  characters,  their  pathogenicity  to 
animals,  and  upon  certain  special  methods  of  investigation. 

Morphology. — A  study  of  the  morphology  of  pathogenic 
organisms  gives  much  important  information,  but  mainly  of 
a  preliminary  kind.  Very  few  bacteria  can  be  even  approxi- 
mately identified  from  their  appearance  alone. 

Micro-organisms  are  primarily  classified  according  to  their 
shape.  A  round  dot  is  a  coccus  or  micro-coccus  ;  a  rod  is  a 
bacillus  ;  a  twisted  spiral  a  spirochete. 

The  shape  may  also  give  more  detailed  information,  since 
some  cocci  are  not  absolutely  round,  but  may  have  their 
opposed  surfaces  flattened  as  in  the  case  of  the  gonococcus, 
or  may  have  pointed  ends  as  the  pneumococcus.  Diphtheria 
bacilli  may  present  racquet  or  club-shaped  extremities  of  a 
very  characteristic  appearance,  while  tubercle  bacilli  stain  in  an 
irregular  or  beaded  pattern. 

Further  information  is  obtained  from  the  grouping  of 
bacteria,  either  as  they  appear  in  the  tissues  or  as  they  are 
obtained  from  culture  media.  Cocci  which  characteristically 
occur  in  pairs,  such  as  the  pneumococcus  or  gonococcus, 
are  classed  as  diplococci,  those  with  a  chain  arrangement  as 
streptococci,  and  those  which  appear  in  groups  or  clusters  as 
staphylococci.  The  bacilli  are  commonly  not  differentiated  in 
this  manner  ;  some  bacilli,  however,  such  as  that  of  anthrax, 
tend  to  grow  in  chains  and  may  be  called  strepto-bacilli. 
Others,  such  as  the  Morax  Axenfeld  bacillus  (the  cause  of  one 


102  CLINICAL   PATHOLOGY. 

variety  of  conjunctivitis),  occur  in  pairs  and  are  known  as 
diplo-bacilli.  The  classification  of  organisms  in  this  manner, 
according  to  their  grouping,  depends  upon  a  knowledge  of  the 
characteristic  behaviour  of  the  bacteria,  and  this  can  only  be 
determined  by  an  investigation  of  their  appearance  in  body 
tissues  as  well  as  by  their  mode  of  growth  in  various  culture 
media.  A  paired  coccus  seen  in  a  film  of  pus  may  be  a 
diplococcus,  a  streptococcus,  or  a  staphylococcus.  It  is 
necessary  to  isolate  the  organism  in  pure  culture  and  examine 
it  when  growing  in  a  liquid  medium  such  as  broth,  and  on  a 
solid  medium  such  as  an  agar  slope.  Diplococci  occur  mainly 
in  pairs  in  all  media.  A  streptococcus  grows  in  long  chains 
in  broth,  and  frequently  only  in  short  chains,  or  even  small 
clumps  on  agar.  Staphylococci  show  their  clump  arrange- 
ment best  on  agar,  and  frequently  appear  in  pairs  or  even  very 
short  chains  in  broth. 

The  size  of  organisms  is  on  the  whole  of  little  assistance 
in  identification,  since  the  same  organisms  growing  under 
favourable  conditions  may  be  very  appreciably  larger  than 
when  the  surroundings  are  unfavourable.  A  colon  bacillus, 
for  example,  may  be  so  short  and  stout  as  to  be  almost 
mistaken  for  a  coccus,  or  it  may  be  long  and  thin.  A  coccus 
may  vary  greatly  in  size,  and  may  in  some  instances,  such  as 
in  the  case  of  the  pneumococcus,  and  more  rarely  the  strepto- 
cocci, be  so  elongated  as  to  resemble  a  bacillus.  At  the  same 
time  with  some  bacilli,  such  as  the  anthrax  bacillus,  the 
individual  members  are  very  commonly  stout  and  long. 

The  motility  or  non-motility  of  organisms  affords  a  further 
clue  to  their  identification,  since  some  organisms,  such  as  the 
diphtheria  bacilli,  have  no  power  of  spontaneous  movement, 
while  other  organisms  are  actively  motile.  The  relative 
motility  of  organisms  in  the  same  group,  such  as  members  of 
the  spirochete  family  and  bacilli  of  the  coli-typhoid  group, 
is  of  considerable  assistance  in  differentiation.  It  must  be 
recognised,  however,  that  different  strains  of  the  same 
organism,  and  even  different  sub-cultures  of  the  same  strain, 
may  show  marked  variability  in  movement.  The  colon 
bacillus,  for  example,  is  as  a  rule  sluggishly  motile,  but  may 
on  occasion  move  as  briskly  as  strains  of  the  more  constantly 
motile  typhoid  bacillus.  The  motility  of  some  organisms 
depends,   in    part    at    least,   on    the    presence    of    flagella, 


INTRODUCTORY— TABLE    OF   CLASSIFICATION.     103 

for  the  demonstration  of  which  special  stains  are  required 
(page  163). 

Capsules  are  present  around  some  organisms,  but  not  others. 
The  presence  of  capsules  may  be  made  use  of  in  identifying 
such  an  organism  as  the  pneumococcus,  which  is  capsulated  in 
preparations  made  from  the  body  tissues,  but  loses  its  capsule 
when  cultivated  on  artificial  media.  The  demonstration  of 
capsules  requires  a  special  method  of  staining  (page  162),  and 
great  caution  must  be  exercised  in  recognising  capsules  in 
films  stained  with  the  ordinary  dyes,  since  there  is  a  tendency 
for  clear  areas  to  appear  in  the  immediate  vicinity  of  the 
organisms  owing  to  a  shrinkage  of  the  fluid  constituents  of  the 
pus  from  the  bacteria,  the  clear  empty  areas  having  the 
appearance  of  capsules.  It  is,  however,  rarely  necessary  to 
investigate  specially  the  presence  or  absence  of  capsules. 

Spores  are  confined  to  certain  bacilli,  and  the  presence  of 
spores  is  of  great  value  in  differentiation,  since  the  majority  of 
pathogenic  bacilli  never  produce  them.  The  spore-bearing 
bacilli  do  not,  however,  always  show  spores,  and  may  do 
so  only  when  grown  in  the  appropriate  media.  The  anthrax 
bacilli  commonly  are  devoid  of  spores  in  preparations  made 
from  the  pustule,  while  in  broth  cultures  spores  only  may  be 
found  and  no  bacilli.  The  situation  of  the  spore  is  of  further 
assistance,  since  it  may  be  central  or,  as  in  the  case  of  the 
tetanus  bacillus,  terminal.  Spores  are  readily  recognised  in 
films  stained  with  the  ordinary  dyes,  and  may  be  further 
identified  by  special  methods  (page  162). 

Staining  properties. — The  staining  properties  of  bacteria 
are  in  effect  micro-chemical  tests  of  their  composition,  and  for 
this  reason  are  of  great  importance  in  their  identification.  The 
majority  of  micro-organisms  stain  with  the  ordinary  dyes, 
such  as  methylene  blue,  carbol  fuchsin,  and  carbol  thionin ; 
the  minority  require  special  staining  processes  for  their 
differentiation.  The  less  important  and  lowly  pathogenic 
mouth  spirochetes,  for  example,  take  fairly  readily  the 
ordinary  stains,  whereas  the  spirochete  of  syphilis  is 
unstained  by  them,  and  can  by  this  method  be  differentiated. 
A  most  important  staining  method  in  use  for  the  identification 
of  organisms  is  that  of  Gram.  Gram's  method  depends  upon 
the  use  of  iodine  as  a  mordant.  All  or  nearly  all  organisms 
are  stained   by  gentian-violet,  and    are  again  decolorised  if 


104  CLINICAL   PATHOLOGY. 

washed  in  alcohol.  If,  however,  the  organisms  after  being 
stained  with  gentian-violet  are  exposed  to  Gram's  iodine 
solution,  the  stain  is  fixed  in  some  organisms  so  as  to  be  no 
longer  dissolved  out  in  the  spirit,  but  not  into  other  organisms. 
Those  bacteria  which  remain  coloured  are  called  "  Gram- 
positive,"  those  which  lose  their  stain  in  the  spirit  are  known 
as  "Gram-negative."  The  majority  of  the  cocci  are  Gram- 
positive,  and  of  the  bacilli  Gram-negative. 

Another  very  important  staining  reaction  is  that  known  as 
the  Ziehl-Neelsen  method,  and  is  used  for  the  identification  of 
the  tubercle  bacillus.  The  tubercle,  leprosy,  and  smegma 
bacilli  can  be  stained  with  hot  carbol  fuchsin,  and  when  thus 
stained  resist  decolorising  with  acids,  whereas  other  organisms 
lose  the  stain  again  when  exposed  to  acids.  The  bacteria  which 
retain  the  stain  are  therefore  known  as  "acid  fast,"  and  can 
by  their  staining  reactions  be  further  differentiated  one  from 
another,  since  the  tubercle  bacillus  retains  the  carbol  fuchsin 
after  exposure  to  25  per  cent,  sulphuric  or  nitric  acid;  the 
leprosy  bacillus  is  decolorised  by  this  strength  of  acid,  but 
remains  coloured  in  12  per  cent,  acid ;  while  the  smegma 
bacillus  is  acid  fast,  but,  unlike  the  tubercle  bacillus,  is  not 
"  alcohol  fast,"  that  is  to  say,  it  gives  up  the  carbol  fuchsin  in 
methylated  spirit. 

Cultural  characters. — The  identification  of  the  great 
majority  of  organisms  necessitates  a  study  of  their  behaviour 
and  mode  of  growth  in  artificial  media  outside  the  body. 
Some  bacteria  do  not  grow  at  all  on  any  media  with  which  we 
are  acquainted  ;  others  require  special  media  and  fail  to  grow 
on  those  in  common  use.  The  majority  grow  readily  on  all 
the  ordinary  media.  To  some  media  substances  are  purposely 
added  which  prevent  the  growth  of  one  class  of  organism  and 
allow  the  growth  of  another  class  ;  bile  salts,  for  example, 
inhibit  the  growth  of  cocci,  but  permit  the  growth  of  bacilli  of 
the  coli-typhoid  group.  The  optimum  temperature  for  the 
growth  of  the  majority  of  pathogenic  organisms  is  that  of  the 
human  body,  namely,  37°  C.  Exceptional  organisms,  such 
as  the  glanders  bacillus,  grow  best  at  a  somewhat  higher 
temperature.  The  temperatures  within  which  bacteria  will 
grow  and  the  temperatures  at  which  they  cease  to  live  form 
a  part  of  the  complete  investigation  of  their  natural  history. 

The  time  taken  for  a  visible  growth  to  appear  in  media  is 


INTRODUCTORY— TABLE    OF   CLASSIFICATION.    105 

usually  from  12  to  24  hours.  A  few  organisms  grow  more 
slowly,  and  in  the  case  of  the  tubercle  bacillus  little  growth  is 
visible  in  less  than  10  days. 

Most  bacteria  are  capable  of  growing  under  both  aerobic  and 
anaerobic  conditions,  but  prefer  the  former.  Very  few  patho- 
genic organisms  are  strict  anaerobes,  the  most  important  being 
the  tetanus  bacillus.  By  a  strict  anaerobe  is  meant  an 
organism  incapable  of  growing  in  the  presence  of  oxygen,  and 
by  an  anaerobic  culture  is  meant  a  culture  tube  placed  in  an 
atmosphere  from  which  the  oxygen  has  been  removed. 

A  brief  account  follows  of  the  more  usual  media  employed 
for  the  cultivation  of  organisms  and  the  changes  which  may 
take  place  in  them  as  the  result  of  bacterial  growth. 

Broth  is  the  most  universal  of  all  media,  and  in  addition 
forms  the  basis  of  numerous  others.  Broth  is  nothing  more 
than  a  solution  of  beef  extract,  with  sodium  chloride  and 
peptone  added.  The  acidity  of  the  original  broth  is  estimated 
and  sufficient  soda  is  added  to  make  the  mixture  alkaline  to 
litmus  and  of  a  certain  acidity  to  phenol-phthalein  (page  181). 

The  majority  of  organisms  growing  in  broth  produce  in  it  a 
general  turbidity,  and  after  a  time  the  deposit  of  a  more  or 
less  felted  mass  of  bacteria  at  the  bottom  of  the  tube.  Some 
organisms,  such  as  the  cholera  vibrio  and  certain  non- 
pathogenic air  bacilli,  form  also  a  thin  pellicle  on  the  surface 
of  the  medium.  The  streptococci,  on  the  other  hand,  leave  the 
bulk  of  the  medium  as  clear  as  it  was  before  inoculation,  and 
form  a  stringy  granular  deposit,  both  floating  free  and  attached 
to  the  sides  of  the  tube. 

These  are  the  appearances  obvious  in  an  inoculated  tube ;  in 
addition  certain  organisms,  such  as  the  colon  bacillus,  have  the 
property  of  producing  indole  from  the  peptone  present  in  the 
medium,  and  this  has  to  be  tested  for  by  adding  yellow  nitric 
acid  (containing  nitrites),  when  a  rose-pink  colour  of  a  nitroso- 
indole  body  diffuses  through  the  medium.  The  cholera  vibrio 
produces  both  indole  and  nitrites,  so  that  the  pink  colour  is 
produced  on  the  addition  of  pure  nitric  or  sulphuric  acid  only, 
a  reaction  which  is  known  as  the  cholera-red  reaction. 

On  examining  therefore  an  inoculated  broth  tube  after 
incubation  one  looks  for  a  general  turbidity  of  the  medium,  a 
pellicle  on  the  surface,  a  deposit  at  the  bottom  or  a  clear 
medium  with   a  granular  deposit  down    the    side,   and  then 


10(5  CLINICAL   PATHOLOGY. 

adds  a  few  drops  of  yellow  nitric  acid  to  test  for  indole 
formation. 

Agar  is  the  most  commonly  used  solid  medium,  just  as 
broth  is  as  a  liquid  medium.  Agar  itself  is  a  carbohydrate 
derived  from  the  stems  of  certain  Chinese  seaweeds,  and  agar 
media  are  prepared  by  adding  this  substance  to  broth.  The 
medium  is  put  up  either  in  the  form  of  "  slope,"  "  stab,"  or 
"  plate "  cultures.  The  slope  culture  tubes  are  made  by 
pouring  a  small  quantity  of  melted  agar  into  a  test  tube,  and 
allowing  it  to  harden  in  a  slanting  position.  The  stab  cultures 
are  tubes  filled  or  partly  filled  with  the  medium,  and  are 
inoculated  by  passing  an  infected  platinum  wire  into  the  heart 
of  the  medium.  They  may  be  used  for  growing  organisms 
under  anaerobic  or  partially  anaerobic  conditions.  The  plate 
cultures  are  made  by  pouring  the  agar  into  flat,  round,  shallow 
dishes,  covered  with  a  loosely  fitting  lid,  and  known  as  Petri 
dishes. 

The  majorhVy  of  organisms  grow  well  on  agar,  and  one  is 
able  to  tell  from  the  type  of  colony  produced  the  class  of 
organism  present.  Bacilli  of  the  coli-typhoid  group  grow  in  a 
continuous  whitish  streak  with  laterally  spreading  edges  up 
the  surface  of  an  agar  slope,  and  in  large  rounded,  opaque 
colonies,  with  thin  crenated  margins  and  heaped-up  centres  on 
plate  cultures.  Staphylococci  produce  large,  round,  opaque 
colonies  with  sharp  edges,  the  colonies  being  white,  lemon- 
coloured  or  yellow  according  to  the  variety  of  staphylococcus 
present.  Streptococci  and  pneumococci  grow  in  tiny  round 
translucent  colonies  barely  visible  to  the  naked  eye.  Gonococci, 
meningococci,  influenza  and  diphtheria  bacilli  are  among  the 
more  important  organisms  which  grow  in  somewhat  similar  so- 
called  "  pin-point  "  colonies.  On  examining  an  inoculated  agar 
culture,  therefore,  one  looks  at  the  type  of  colonies  present, 
whether  they  are  large  or  small,  white  or  coloured,  translucent 
or  opaque,  whether  the  margins  are  rounded  or  crenated,  and 
whether  the  colonies  are  discrete  or  grown  together  to  form  a 
continuous  streak.  In  the  case  of  stab  cultures  one  looks  to 
see  if  the  growth  is  more  abundant  at  the  surface  where  there 
is  more  oxygen,  or  in  the  depths  of  the  medium  where  oxygen  is 
scanty  and  anaerobic  bacteria  grow  more  readily. 

Gelatin  media  consist  of  broth  with  sufficient  gelatin 
added  to  produce  a  solid  medium  at  room  temperature.     Since 


INTRODUCTORY— TABLE    OF   CLASSIFICATION.    107 

gelatin  media  are  liquefied  at  37°  C.  it  is  necessary  to  incubate 
the  tubes  at  or  a  little  above  room  temperature,  that  is  from 
18  to  22°  C.  Gelatin  is  put  up  in  slope,  stab  and  plate  cultures 
in  the  same  manner  as  agar.  The  most  important  feature  of 
gelatin  as  a  medium  is  the  fact  that  some  organisms  in  their 
growth  are  able  to  liquefy  it,  while  others  do  not.  The  coli- 
typhoid  group  of  bacilli  do  not  liquefy  gelatin,  and  can  thus  be 
differentiated  from  such  bacilli  as  proteus  and  pyocyaneus, 
which  do.  Some  organisms,  such  as  the  pneumococci,  which 
are  capable  of  growing  on  the  majority  of  the  usual  media,  do 
not  grow  on  gelatin.  In  stab  cultures  a  few  bacteria,  for 
example  anthrax  bacilli,  send  out  characteristic  lateral 
processes  radiating  from  the  line  of  the  stab.  A  gelatin 
medium,  therefore,  should  be  incubated  at  a  relatively  low 
temperature  and  should  be  examined  for  the  presence  and 
nature  of  the  growth,  and  in  particular  for  the  presence  or 
absence  of  liquefaction. 

Litmus  milk  consists  of  fresh  sterilised  milk  rendered 
slightly  alkaline  with  soda  and  coloured  blue  with  litmus 
solution.  Litmus  milk  is  a  most  valuable  medium,  partly 
because  the  majority  of  organisms  grow  in  it  abundantly  and 
partly  because  of  the  various  changes  which  they  may  set  up 
in  it  and  by  which  they  may  be  differentiated.  Organisms 
growing  in  milk  may  produce  no  change  in  it  or  may  render  it 
more  alkaline  or,  more  commonly,  may  either  simply  produce 
an  acid  reaction  in  it  or  both  acidify  and  clot  it.  Other 
organisms  may  not  only  acidify  and  clot  the  milk,  but  may 
eventually  decolorise  the  litmus  and,  further,  may  peptonise 
the  clot  and  render  it  liquid  again.  The  bacillus  enteritidis 
sporogenes  first  clots  the  milk,  and  then  by  virtue  of  the  gas 
produced  by  it  blows  the  curd  up  the  sides  of  the  tube,  leaving 
the  whey  at  the  bottom.  Litmus  milk,  therefore,  may  be 
unchanged,  or  may  be  rendered  more  alkaline,  or  may  be 
acidified  and  remain  liquid,  or  may  be  both  acidified  and 
clotted. 

Litmus  carbohydrate  broth. — A  series  of  media  may  be 
made  with  broth  to  which  litmus  is  added  and  1  per  cent,  of 
a  variety  of  carbohydrates.  The  purpose  of  these  media  is  to 
test  the  capability  of  organisms  to  break  up  the  carbohydrates 
present  and  to  produce  from  them  an  acid  or  an  acid  and  gas. 
The  production  of  acid  is  shown  by  the  change  of  colour  of  the 


108  CLINICAL  PATHOLOGY. 

litmus  medium  from  blue  to  red.  In  order  to  observe  the 
formation  of  gas  it  is  convenient  to  fill  a  small  tube  with  the 
medium  and  sink  it  upside  down  in  the  culture  tube  so  that 
the  gas  may  collect  in  the  small  inverted  tube.  Numerous 
carbohydrates  are  employed  in  making  the  media ;  those  in 
fairly  common  use  are  glucose,  lactose,  mannite,  salicin  and 
raffinose.  In  examining  the  media  after  inoculation  look  for 
the  same  changes  that  are  to  be  found  in  ordinary  broth,  such 
as  the  presence  of  a  general  turbidity  or  of  a  clear  medium 
with  a  granular  deposit,  and  in  addition  observe  the  colour  of 
the  litmus  and  the  presence  or  absence  of  gas  in  the  small 
inverted  tube.  The  production  of  an  acid  reaction  is  not 
necessarily  accompanied  by  the  evolution  of  gas. 

Neutral  red  broth  consists  of  broth  with  a  dye  called 
neutral  red  added.  The  medium  is  a  useful  one  for  distinguish- 
ing organisms,  and  particularly  bacilli  of  the  coli-typhoid 
group.  The  colon  bacillus  alters  the  red  colour  of  the  broth 
to  a  distinct  yellow  and  also  produces  in  the  medium  a  green 
fluorescence ;  the  typhoid  bacillus  produces  no  change  other 
than  the  general  turbidity  of  its  growth. 

MacConkey's  bile  salt  medium. — This  is  a  solid  medium 
commonly  used  in  plate  cultures  for  the  purpose  of  isolating 
colonies  of  the  coli-typhoid  group  from  mixtures  of  organisms. 
The  medium  has  as  its  basis  agar,  and  in  addition  sodium- 
taurocholate,  lactose  and  neutral  red.  The  presence  of  the 
bile  salts  inhibits  the  growth  of  the  great  majority  of  organisms 
other  than  members  of  this  group.  The  neutral  red  gives  a 
different  colour  reaction  with  those  organisms  which  ferment 
the  lactose  to  those  which  do  not.  The  medium  is  there- 
fore extremely  useful  for  the  isolation  of  bacilli  from  such 
sources  as  the  urine  and  fseces,  since  the  cocci  and  other  non- 
pathogenic bacteria  do  not  grow  on  it,  while  the  colon  bacillus 
grows  in  bright  red,  and  the  typhoid  bacillus  in  whitish  yellow 
colonies. 

Special  media. — While  the  above  media  are  commonly 
used  for  organisms  of  ready  growth,  a  minority  of  pathogenic 
bacteria  do  not  grow  at  all  on  them,  or  grow  very  poorly. 
Numerous  special  media  have  been  devised  for  such  organisms, 
and  a  few  are  in  common  use.  Blood  serum  media  are  essential 
for  the  growth  of  some  bacteria,  of  which  the  most  important 
is  the  gonococcus.     The  blood  serum  can  be  used  alone  in  the 


INTRODUCTORY— TABLE    OF    CLASSIFICATION.    109 

form  of  slope  cultures  after  inspissating  the  serum  in  steam, 
or  it  may  be  mixed  with  agar.  The  simplest  medium  of  this 
nature  is  made  by  smearing  a  drop  of  sterile  blood  over  the 
surface  of  an  agar  slope.  A  valuable  medium  also  is  "  Nasgar," 
which  consists  of  nutrose,  ascitic  fluid  (or  blood  serum)  and 
agar. 

The  essential  elements  of  the  blood  serum  may  be  derived 
from  a  variety  of  sources,  such  as  hydroceles,  ovarian  cysts 
and  ascitic  fluids.  The  serum  is  most  readily  obtained  from  a 
large  animal,  such  as  an  ox  or  a  sheep,  and  for  the  purpose  of 
growing  the  diphtheria  bacillus  the  former  source  is  prefer- 
able, since  this  bacillus,  whose  growth  on  ordinary  agar  is 
apt  to  be  crowded  out  by  the  growth  of  other  organisms, 
multiplies  rapidly  on  ox  serum  and  outgrows  the  common 
cocci.  The  tubercle  bacillus  is  an  example  of  another 
organism  which  will  not  grow  on  the  ordinary  media,  but  will 
grow  in  broth  or  on  agar  to  which  glycerin  has  been  added. 
Another  useful  medium  for  the  growth  of  the  tubercle  bacillus 
is  made  from  eggs,  and  is  perhaps  the  most  commonly  used 
medium  for  this  purpose :  both  the  white  and  the  yolk  of  the 
egg  are  used  and  slope  cultures  of  the  solidified  medium  are 
put  up.  Another  special  medium  may  be  mentioned,  consist- 
ing of  agar  to  which  glycerin  and  oleic  acid  have  been  added, 
and  which  is  used  for  the  cultivation  of  the  acne  bacillus. 

Animal  inoculation. — Animal  inoculation  is  resorted  to 
for  purposes  of  testing  the  pathogenicity  of  an  organism, 
or  for  isolating  it,  or  for  determining  its  nature.  The  animals 
most  commonly  employed  are  guinea-pigs,  mice  and  rabbits. 
A  few  examples  only  may  be  given  here  of  the  occasions  on 
which  animals  should  be  used.  In  suspected  cases  of  diphtheria 
organisms  may  be  isolated  which  exactly  resemble  the  genuine 
bacillus  in  appearance,  in  staining  reactions  and  in  cultural 
characters,  yet  may  differ  from  it  in  the  very  important 
circumstance  that  they  are  non-virulent  and  produce  no 
toxin.  In  cases  of  doubt  the  only  reliable  test  is  to  inoculate 
a  guinea-pig  with  a  suspension  of  the  bacteria.  The  innocuous 
or  so-called  diphtheroid  bacilli  do  not  affect  the  animal ;  the 
genuine  diphtheria  bacilli  kill  it  within  36  hours.  The  test 
may  be  further  elaborated  by  inoculating  a  second  animal 
with  the  bacilli  after  previous  injection  of  anti-diphtheritic 
serum,  no  ill  effect  resulting.     The  virulence  of  streptococci 


110  CLINICAL   PATHOLOGY. 

or  pneumococci  is  best  tested  on  mice  or  rabbits,  since 
these  animals  are  more  susceptible  than  guinea-pigs.  The 
identification  of  the  tetanus  bacillus  is  completed  by  the 
production  of  tetanic  spasms  following  the  inoculation  of  the 
suspected  organism  in  mice.  The  glanders  bacillus  is  rarely 
met  with  in  human  pathology,  and  its  identity  should  be  con- 
firmed by  the  intra-peritoneal  inoculation  of  a  male  guinea- 
pig,  a  characteristic  suppuration  resulting  in  the  tunica 
vaginalis.  Body  fluids  suspected  to  be  tuberculous,  yet  in 
which  no  tubercle  bacilli  can  be  detected  microscopically  or  by 
cultures,  may  be  proved  to  be  infected  by  means  of  guinea-pig 
inoculation.  The  guinea-pig  is  highly  susceptible  to  the 
human  tubercle  bacillus,  and  the  smallest  dose  injected  into 
the  leg  leads  to  tuberculosis  of  the  nearest  lymphatic  gland 
within  10  days,  and  to  death  from  generalised  tuberculosis  in 
from  4  to  6  weeks.  The  tubercle  bacillus  can  be  identified 
and,  if  necessary,  recovered  in  culture  from  the  lesions  in  the 
guinea-pig. 

Special  Methods. — Certain  other  methods  of  identifying 
organisms  have  been  described  in  the  section  on  the  blood. 
These  include  the  agglutination  tests,  in  which  a  known  serum 
is  mixed  with  a  suspension  of  an  unknown  bacillus,  a  method 
most  frequently  used  for  the  rapid  identification  of  the  typhoid 
bacillus.  A  similar  method,  less  commonly  employed,  is  the 
complement  deviation  test  as  elaborated  by  Bordet  and 
Gengou. 

Another  method,  which  is  used  for  the  identification  of  the 
cholera  vibrio,  is  that  known  as  Pfeiffer's  reaction.  A  mixture 
is  made  of  a  suspension  in  broth  of  the  organism  to  be  tested 
and  anti-cholera  serum  and  the  mixture  is  injected  into  the 
peritoneal  cavity  of  a  guinea-pig.  After  a  few  minutes  the 
peritoneal  fluid  is  examined,  and  if  the  spirilla  injected  were 
cholera  spirilla  they  are  found  to  lose  their  motility,  break  up 
into  globules  and  disappear ;  if  they  were  not  cholera  spirilla 
they  are  found  to  be  unaffected.  A  control  experiment  in 
which  normal  serum  replaces  the  anti-cholera  serum  should 
always  be  performed  upon  another  animal.  The  reaction  can 
also  be  made  in  vitro  by  mixing  the  vibrios,  the  anti-cholera 
serum  and  fresh  guinea-pig  serum,  and  incubating  in  the  form 
of  a  hanging  drop  for  from  1  to  2  hours. 

If  bacteria,  isolated  and  identified  by  the  methods  indicated 


INTRODUCTORY— TABLE   OF   CLASSIFICATION.     Ill 

above,  are  classed  primarily  on  the  basis  of  their  morphology 
and  staining  reactions,  and  secondarily  according  to  their 
cultural  characters,  they  will  be  found  to  fall  into  groups  of 
which  the  individual  members  resemble  each  other,  not  only  in 
their  laboratory  characteristics,  but  also  in  the  nature  of  the 
lesions  which  they  produce  in  the  human  subject. 

In  the  accompanying  table  the  pathogenic  bacteria  are 
divided  into  cocci,  bacilli,  spirilla,  streptothrices,  etc.,  and  sub- 
divided into  those  which  are  Gram-positive  or  Gram-negative, 
acid  fast  or  non-acid  fast.  The  Gram-negative  bacilli  are  further 
sub-divided  into  groups  according  to  their  cultural  characters. 
It  is  not  intended  that  a  table  condensed  into  a  page  should 
replace  the  larger  text-books  of  bacteriology,  but  it  is  con- 
venient for  the  student  to  have  some  scheme  into  which  he 
can  work  the  more  detailed  knowledge  subsequently  acquired. 
In  many  excellent  bacteriological  text-books  organisms  closely 
allied  in  their  staining  and  cultural  properties  may  be  widely 
separated  in  the  text,  with  the  disadvantage  that  all  the 
methods  by  which  the  bacteria  have  to  be  identified  must  be 
learned  for  each  organism  instead  of  for  each  group  of 
organisms.  The  most  characteristic  special  reactions  for  the 
identification  of  the  individual  members  of  the  groups  are 
further  indicated  in  the  table,  an  amplification  of  which  forms 
the  basis  of  the  following  chapters. 

The  division  of  bacteria  into  groups  in  this  manner 
has  certain  justifications  other  than  convenience,  since  it 
indicates  to  some  extent  the  origin  of  bacterial  species. 
There  can  be  little  doubt  that  at  one  period  of  the  world's 
history  all  pathogenic  bacteria  were  saprophytes,  that  is  to 
say,  lived  upon  their  hosts  without  harming  them  and  pro- 
duced no  highly  specialised  and  deadly  toxin.  To  each  group 
of  pathogenic  bacteria  in  the  table  could  be  added  a  non- 
pathogenic prototype  closely  resembling  the  other  members  of 
the  group  in  morphological,  staining  and  cultural  characters. 
As  examples  may  be  given  the  practically  non-pathogenic 
staphylococcus  albus  of  the  skin  among  the  Gram-positive 
cocci,  the  hay  and  butter  bacilli  of  the  acid-fast  group,  the 
diphtheroid  bacilli,  and  the  harmless  mouth  spirochetes. 

It  must  not  be  supposed  that  the  acquirement  of  toxins  by 
bacteria  has  been  directed  solely  against  humanity.  From  the 
same   non-pathogenic  prototype  can   often   be    traced    those 


•ranias  pocqq  Xji^x 
-noi;.XBd  'mpain  p3ioads 
.13}3jd  ptre  pooo  + 
otbi-q  aq^  uuq;  jfpttojs 
9.10m  a\oj§  :  dd\\ 
pjooooo^dans  30  s9iuo[oo 


CD  ^ 

ass. 


."  -         PI 


p  c   y 
5 


03  T4  _ 
C    03 


»    «*  ri 

^    B  S 

2^  5 

c5 


rt 


c 


S9}uip£q 

-OqXBO  A\3J  ^JipiOB 

saraojoo  ^niod 
-uid    ^naoiqsnuiq. 


O 

O 


CO  /2 

'  5 'S 


Pi  ^ 


~     ^     QJ     ,  ,     ^     r* 

O  ^  i>»*a  *  >> 
oj  £  a>  "8  •— 

m  "o 

to  03 


Pi 

o 
o 

S-. 


'rt  ^  n 


CO     DO 

SO 


j7,    D    3    B 

o  P?  —  o> 

"2 -2*  a  a 


+ 


P4 


A 


<a< 


■a 


o  o  g 


i- 

0 

-J 

<D 

— 

!*r^? 

gf.2 
P-  -/.  — 


1i  <u 

o  H  c 

ic  q  S 

CO     ^  CO 

<u   y  a> 

o  £  o 

ci  T3  c3 


I— I 

o 


cS 

J-l 

O 
^ 

2 

&o 

B 

03 

+J 

CO 

a 

CO 

0)    <u 

"3 

e3 

CD  .H 

0    O 

O  £ 

o  a. 

^J 

43 

a 

CO 

03 

O    X 

CD  'o 

°  88 

B 
&l 

CO 
CD 

a 

c  ° 

O   to 
co  -t: 

.2  0 

CO 

c 

B 

ao 

.«  a* 

O  T 

n^ 

+-> 

v.  ^  a 

c 

O    <D 

oJ 

~t  -2    0 

O  " 

B 

■2?   &  OS 

00  co 

&.  0 ,— 1 

to 

— "— v—  & 


ffi 


v. 


ii  x 

18   »   g 

nneu 
in  d 

otile. 

u    6J3  £. 
3  'O  J3 
3   d   0 

•  rH     m       h-C     Ij^ 

O    6C. , 

seen 
and 
our 
ag- 

■  +3 

••  0    : 

Stj  o^3 

•    S-,       '• 

0-30 

S   j         co 

:  *  o, 

»        j;  0 

:      ^2 

e. 
th. 
ores 
icti 

■2     CO     g 

«  ?>  32 

•S  0  a-o 

0  n  « 

ROci 

Oh 

£           & 

cTQ 

to 

ES 

4^ 

d> 

a     : 

roteus 
pocyan 

<S     • 

a  cs 

.spa 

PM 

P4 

SB 

2  3 

0  :t 

0 

3 

yellow  cols. 

red  cols. 
- 

k 

CM 

1 

nil 
a+gas 

nil 

acid 

a+gas 

O 

0 

U 

y, 

CO 

Q 

CO                                                         </, 

s  be       -a    -  §, 

2  4-                           u                 _L 

0 

CS 

1-^ 

CO 

^SCS        -       -IS        _     60 

s  s     ~     -    s     ~  +     - 

no  change 
yellow 

no  change 

yellow  + 
fluorescence 

CD 
O 

■a 

1    1      1      1      I      1     + 

5»! 

S 

acid 

acid 

il  then 

alkaline 

a  then 
alkaline 

a + clot 

a   1 

•< 

Thin  delicate  streak  to 
opaque  growth  with 
spreading  edges — > 

M 


hi 
hi 
1— 1 

Ph 


"lO^Sfflfi0!! 


_C0    CD    ^  ^    co 


c3 


rf-5  a 


.w    op."  _« 
"^    CO    CD    CD    i-Ml-, 

'Ebo       .    cd  ^ 


3      .„ 


o 
W 
Ph 

w 


3  <* .3  js 


it  11 

3  y  o 


O   O   o  ^  _^  ~- 

g  K02  ^  3  ^ 

go-go  ».g 
s->  -5  o  o  ST-a 
•S2  -2  -o  o  -^  S 


^  g 


H      L,      ^      (O 

08  -rt    to  j£ 

Ph  P4  <J  gH 


.   +  3 
o 

I— I 
Pn 

o 

EH 
Ph 

03         CO  .—I     CD 

JT^     ~  eS  ■- 

g    CD    o  ^< 

S  J39        ?! 

Q     c<5    to    cj  oj 


c3   > 


114  CLINICAL   PATHOLOGY. 

organisms  which  cause  similar  diseases  in  animals,  and  which 
have  retained  many  of  the  characteristics  of  the  group  they 
belong  to.  For  example,  the  gonococcus  will  only  live  and 
produce  disease  in  the  human  subject,  but  there  is  a  contagious 
urethritis  found  in  bulls  producing  the  so-called  granular 
vaginitis  of  cows,  of  which  the  causative  organism  is  a  coccus 
and  Gram-negative.  The  tubercle  bacilli  are  also  distinctive 
for  man,  bovines,  birds  and  fish.  Although  these  allied 
bacteria  closely  resemble  each  other  in  so  far  as  our  com- 
paratively crude  methods  permit  investigation,  it  must  be 
recognised  that  each  member  is  a  distinct  and  established 
species.  The  rapid  multiplication  of  micro-organisms  might 
lead  us  to  expect  a  rapid  variation  of  species,  but  since 
bacteria  multiply  by  the  simple  process  of  splitting  in  two 
the  minimal  opportunity  for  variation  exists,  and  such  varia- 
tion as  does  take  place  is  largely  evolved  out  of  minute 
alterations  in  the  composition  of  the  bacterial  toxin.  Con- 
sequently we  should  not  expect  to  change  human  tubercle 
bacilli  into  the  bovine  or  avian  species  by  passing  them 
through  numerous  members  of  the  appropriate  animals  over 
intervals  of  a  few  years,  and  as  a  fact  the  change  does  not 
occur.  The  establishment  of  species  has  taken  place  by 
natural  selection  in  the  course  of  ages. 

It  is  well  to  recognise  also  that  the  most  virulent  of  human 
bacteria  are  still  capable  of  maintaining  in  the  tissues  a  harm- 
less saprophytic  existence.  The  pneumococcus  may  and  often 
does  exist  in  the  mouth,  and  less  frequently  the  typhoid 
bacillus '  in  the  gall  bladder,  without  causing  disease.  The 
presence  of  the  essential  organism  is  not  the  only  factor  in  the 
production  of  the  disease,  and  it  should  be  evident  that  the 
isolation  of  bacteria  from  the  body  can  have  little  meaning  or 
significance  apart  from  the  clinical  investigation  of  the  nature 
of  the  lesion  present. 


PLATE    VIII. 


>*       * 

s 

7 

! 

PLATE    VIII. 


Staphylococci. 

In  Pus  from  Abscess  of  Neck. 
(Carbol-thionin.) 


Streptococci. 

In  Pus  from  a  case  of  Cellulitis. 
(Carbol-thionin . ) 


Gonococci. 

From  a  Urethral  Discharge. 
(Carbol-thionin.) 


Tubercle  Bacilli. 

In  Sputum. 
(Carbol  Fuchsin  and  Methylene  Blue.) 


Diphtheria  Bacilli. 

Culture  on  Blood  Serum. 
(Loffler's  Methylene  Blue.) 


Anthrax  Bacilli. 

Culture  on  Gelatin. 
(Ccubol-thionin.) 


Tetanus  Bacilli. 

Anaerobic  Culture  on  Agar. 

(Carbol  Fuchsin  and  Methylene  Blue.) 


Influenza  Bacilli. 

Tn  Pus  from  Knee  Joint. 
(Carbol  Fuchsin.) 


CHAPTER  IX. 

the  cocci — the  gram-positive  bacilli. 

The  Gram-positive  Cocci. 

This  group  of  organisms  comprises  the  common  pyogenic 
cocci,  and  gives  rise  to  a  number  of  morbid  conditions  which 
have  in  common  a  comparatively  brief  incubation  period, 
an  acute  onset,  the  local  production  of  pus,  a  usually  rapid 
recovery,  and  a'  tendency  to  leave  the  patient  predisposed  to 
future  attacks. 

The  Gram-positive  cocci  include  the  staphylococci,  the 
pneumococcus,  and  the  streptococci. 

The  staphylococci  (Plate  VIII.). — The  staphylococci  grow 
in  groups  or  clusters,  an  arrangement  best  seen  in  films  pre- 
pared from  solid  media  and  least  obvious  in  broth  culture. 
In  preparations  made  from  pus  the  organisms  are  usually  in 
pairs  with  occasional  clumps,  and  are  readily  taken  up  by  the 
phagocytes.  It  is  a  common  elementary  error  to  suppose  that 
numerous  diplococci  within  a  polynuclear  cell  are  necessarily 
gonococci. 

On  slope  or  plate  cultures  the  staphylococci  grow  in  large, 
round,  opaque,  discrete  colonies  with  clean-cut  edges.  In 
broth  they  produce  a  general  turbidity.  Litmus  milk  is 
usually  acidified  and  clotted.  Gelatin  is  liquefied,  and  a  green 
fluorescence  is  produced  in  neutral  red  broth.  The  majority 
of  the  litmus  carbohydrate  media  are  acidified,  but  no  gas  is 
produced  by  these  or  any  other  cocci. 

These  organisms  therefore  as  a  group  grow  readily  in  all 
the  usual  media  and  produce  active  changes  in  them.  The 
staphylococci  are  divided,  according  to  the  nature  of  the 
pigment  they  produce  when  growing  on  solid  media,  into 
S.  aureus,  S.  citreus,  S.  dibits.  The  colour  of  the  pigment  is 
a  fairly  constant  feature,  and  is  most  obvious  in  a  recently 
isolated  coccus,  but  may  not  appear  until  the  culture  medium 
has  been   exposed  to   direct  sunlight  for  some  hours.     The 


116  CLINICAL   PATHOLOGY. 

power  of  pigment  production  may  be  lost  in  old  cultures,  and 
it  is  probable  that  there  is  little  racial  difference  between  the 
varieties  of  staphylococci  obtained  from  human  lesions.  As  a 
general  rule,  S.  aureus  is  the  most  virulent  of  the  staphylo- 
cocci and  the  most  active  in  culture  media.  S.  citreus 
occupies  an  intermediate  position  and  is  comparatively  seldom 
met  with.  S.  albus  is  usually  the  least  virulent  and  the  least 
active  in  culture  media.  White  staphylococci  are  frequently 
obtained  from  the  skin  which  are  practically  non-pathogenic 
and  very  inactive  in  the  various  media,  often  producing  no 
change  in  litmus  milk. 

The  most  characteristic  lesion  produced  by  the  staphylococci 
is  the  formation  of  a  local  abscess.  The  most  virulent  lesion 
is  the  acute  suppurative  epiphysitis  or  osteomyelitis  of  children, 
the  causative  organism  being  commonly  S.  aureus  and  less 
frequently  a  streptococcus,  pneumococcus,  or  one  of  the  other 
staphylococci.  The  staphylococci  have  a  particular  affinity 
for  the  lymphatic  tissues,  and  often  give  rise  to  metastatic 
abscesses  in  the  lymph  glands.  The  condition  known  as 
lymphangitis  is  practically  always  produced  by  S.  aureus. 
Boils,  carbuncles,  and  the  suppurative  lesions  engrafted  upon 
acne  are  among  the  local  conditions  caused  by  staphylococci. 
The  organisms  may  further  be  spread  from  a  local  source  by 
the  blood- stream  and  give  rise  to  abscesses  in  the  tissues  and 
joints,  a  condition  known  as  pyaemia. 

The  staphylococci  are  also  very  commonly  found  as  secondary 
invaders  of  tissues  affected  by  some  other  agent,  and  particularly 
by  the  tubercle  bacillus. 

The  pneumococcus  (Frankel's  diplococcus :  Diplococcus 
lanceolatus)  (Plate  X.). — The  pneumococcus  occupies  an 
intermediate  position  between  the  staphylococci  and  strep- 
tococci. It  is  an  encapsulated  diplococcus,  which  loses  its 
capsule  in  cultures  but  regains  it  after  inoculation  into  an 
animal.  The  coccus  is  commonly  not  rounded,  but  shaped 
like  the  triangular  blade  of  a  spear,  the  bases  of  the  triangles 
being  opposed  to  each  other  in  a  single  pair.  Not  infrequently 
one  member  of  the  pair  is  lance-shaped  and  the  other  round. 
In  preparations  made  from  liquid  media  the  organisms  may 
be  found  in  short  chains  of  4  or  6  members,  but  the  great 
majority  are  in  pairs.  In  films  of  pus  the  diplococci  are 
sometimes   found   agglutinated   into   clumps   of   considerable 


THE   COCCI— THE   GRAM-POSITIVE   BACILLI.     117 

size.      It  is   extremely  rare   to  find   them  taken  up  by  the 
phagocytes  in  any  numbers. 

The  appearance  of  Gram-positive,  extra-cellular,  encapsulated, 
lance-shaped  diplococci  in  pus  is  very  suggestive  of  the 
pneumococcus  ;  but  the  diagnosis  of  the  organism  should  never 
rest  upon  film  preparations  alone,  since  innumerable  mistakes 
have  thus  been  made.  The  cultural  characters  must  be 
investigated  also.  The  organism  grows  fairly  readily  on 
the  ordinary  media,  but  seldom  so  freely  as  the  other  members 
of  this  group.  It  grows  best  on  agar  or  serum  agar  and  in  milk. 
It  does  not  grow  on  gelatin.  On  agar  it  produces  minute 
round  translucent  colonies.  Milk  is  acidified  and  usually 
clotted  after  2  to  3  days.  A  general  turbidity,  together  with 
a  less  obvious  granular  deposit,  is  produced  in  broth.  Few 
only  of  the  carbohydrate  media  are  acidified,  the  most  constant 
being  dextrose  and  raffinose. 

If  the  pneumococcus  is  inoculated  subcutaneously  into  a 
mouse  the  animal  dies  of  a  rapid  septicaemia,  and  films  made 
from  the  heart  blood  after  death  are  found  to  be  swarming 
with  diplococci. 

The  most  characteristic  lesion  produced  by  the  pneumococcus 
is  lobar  pneumonia,  and  the  organism  is  found  in  the  sputum, 
the  lung  tissue,  and  the  blood.  The  common  comiDlication 
of  pneumonia,  an  empyema,  is  almost  always  due  to  the 
pneumococcus,  which  can  be  readily  demonstrated  in  the  pus. 
The  organism  may  also  produce  an  arthritis  (usually  of  the 
large  joints),  a  peritonitis,  a  salpingitis  (particularly  in  young 
girls),  and  a  meningitis.  Pneumococcal  arthritis  and  peritonitis 
may  arise  as  sequels  of  lobar  pneumonia,  or  much  more 
commonly  independently  of  it.  These  affections  are  more 
frequent  in  children  than  in  adults,  often  give  rise  to  com- 
paratively little  general  disturbance,  and  are  of  favourable 
prognosis.  Pneumococcal  meningitis  is  a  somewhat  rare 
affection,  is  usually  secondary  to  suppuration  in  the  middle 
ear,  and  runs  a  rapidly  fatal  course.  The  pneumococcus  is  a 
rare  cause  of  conjunctivitis,  but  is  often  associated  with 
serpiginous  ulcer  of  the  cornea.  A  particularly  virulent  form 
of  infective  endocarditis  may  be  set  up  by  the  pneumococcus, 
usually  as  a  sequel  of  lobar  pneumonia,  but  it  is  a  rare  com- 
plication. The  organism  may  be  found  in  the  oral  cavity  of 
healthy  persons,  and  can  frequently  be  isolated  from  the  sputum 


118  CLINICAL  PATHOLOGY. 

in  cases  other  than  lobar  pneumonia.  A  diagnosis  of  pneu- 
monia from  the  bacteriological  examination  of  the  sputum  is 
most  unsatisfactory. 

The  streptococci  (Plate  VIII.).— The  streptococci  grow 
in  chains,  an  arrangement  best  seen  in  liquid  media.  The 
number  of  cocci  in  a  chain  varies  from  10  to  over  100.  In 
preparations  made  from  pus  short  chains  are  usually  numerous, 
but  the  majority  of  the  organisms  may  be  in  pairs,  and  many 
of  them  are  found  within  the  phagocytes.  In  cultures  some 
of  the  cocci  may  show  elongated  bacillary  forms.  On  solid 
media  the  streptococci  grow  in  small  round  translucent  "  pin- 
point "  colonies.  Broth  shows  a  granular  deposit  on  the 
sides  of  the  tube  with  a  few  white  granular  colonies  floating 
in  an  otherwise  clear  medium.  Gelatin  is  not  liquefied,  and 
as  a  rule  few  of  the  carbohydrate  media  are  acidified.  There 
are  in  all  probability  a  considerable  number  of  different  types 
of  streptococci,  the  complete  differentiation  of  which  is  beyond 
the  scope  of  this  book.  In  human  pathology  the  most 
important  type,  and  the  one  most  frequently  met  with,  is  the 
Streptococcus  pyogenes.  Two  other  less  constant  types  of 
streptococci  have  been  named  S.  salivarius  and  S.fcecalis. 

The  >S'.  pyogenes  aj)pears  as  a  rule  in  moderately  long 
chains  of  30  to  40  cocci.  Litmus  milk  is  acidified,  but  never 
clotted.  Neutral  red  broth  in  anoerobic  culture  is  unaltered. 
Lactose,  dextrose,  and  salicin  are  acidified.  Inoculation  into  a 
mouse  is  followed  by  rapid  septicaemia  and  death. 

The  most  characteristic  lesions  produced  by  this  organism 
in  man  are  the  acute  spreading  inflammations  of  the  nature  of 
cellulitis.  Erysipelas,  which  at  one  time  was  considered  to  be 
caused  by  a  special  organism,  the  Streptococcus  erysipelatis, 
may  be  due  to  any  of  the  streptococci  and  very  rarely  to  the 
pneumococcus ;  by  far  the  most  common  causative  organism 
is,  however,  S.  pyogenes.  The  difference  between  erysipelas 
and  cellulitis  is  largely  one  of  situation,  the  former  condition 
being  an  acute  inflammation  of  the  skin,  the  latter  of  the 
subcutaneous  tissues.  S.  pyogenes  may  in  addition  cause  acute 
inflammation  in  almost  any  part  of  the  body,  and  the  infec- 
tions set  up  by  it  are  usually  of  a  serious  nature.  The  lymph 
glands  present  a  very  feeble  barrier  to  the  spread  of  the 
organisms,  which  not  infrequently  gain  entrance  into  the 
general  circulation,  and  may  be  recovered  from  it  in  blood 


THE   COCCI— THE   GRAM-POSITIVE   BACILLI.     119 

cultures.  Puerperal  septicaemia  is  a  typical  example  of  local 
infection  by  the  S.  pyogenes  with  a  general  dissemination  in 
the  blood-stream.  Infective  endocarditis  in  the  acute  and 
virulent  variety  which  follows  a  local  infection  is  commonly 
produced  by  this  organism. 

S:  salivarius,  or,  as  it  is  sometimes  called,  S.  brevis,  appears 
as  a  rule  in  short  chains  of  8  to  12  members.  Litmus  milk 
is  acidified  and  usually  clotted.  A  green  fluorescence  is  often 
produced  in  neutral  red  broth  under  anaerobic  conditions.  The 
coccus  is  non-pathogenic  to  mice. 

S.  salivarius  is  commonly  present  in  the  normal  saliva,  and 
a  closely  allied  type  known  as  S.  anginosus,  which  grows  in 
longer  chains,  is  a  frequent  inhabitant  of  the  throat  and  very 
constantly  present  in  the  angina  of  scarlet  fever.  S.  Salivarius 
is  found  in  lesions  of  a  usually  milder  type  than  those 
associated  with  S.  pyogenes.  It  is  the  type  of  organism 
most  frequently  obtained  in  blood  cultures  from  cases  of 
chronic  infective  endocarditis,  such  as  result  from  old 
rheumatic  infections.  The  organism  known  as  the  Diplo- 
eoccus  rheumaticus,  and  believed  by  some  to  be  the  cause 
of  acute  rheumatic  fever,  is  probably  identical  with 
S.  salivarius.  This  streptococcus  is  also  often  associated 
with  severe  pyorrhoea  alveolaris,  and  may  be  a  factor  in  the 
production  of  some  toxic  lesions  which  accompany  an  infective 
condition  of  the  mouth,  particularly  the  variety  of  osteo- 
arthritis which  mainly  affects  the  hands  and  is  so  fre- 
quently preceded  by  a  local  inflammatory  lesion  in  the  mouth 
or  elsewhere. 

S.fiecalis  grows  in  short  chains,  clots  milk,  alters  neutral 
red  broth,  and  acidifies  the  majority  of  the  carbohydrate 
media,  including  litmus  mannite.  It  is  non-pathogenic  to 
mice.  The  organism  is  commonly  present  in  the  fasces,  but 
is  not  found  with  any  frequency  in  human  lesions.  It  may 
be  isolated  from  sinuses  or  from  spreading  inflammations  in 
association  with  the  gut,  and  is  found  less  commonly  in  cases 
of  infective  endocarditis. 

The  Gram-negative  Cocci. 

The  organisms  of  this  group  present  certain  features  in 
common.  The  cocci  grow  slowly  and  with  difficulty  or 
not    at     all    on    the    ordinary    media.       They     grow    well 


120  CLINICAL  PATHOLOGY. 

on  media  containing  blood.  The  colonies  produced  on  solid 
media  are  of  the  streptococcal  type.  In  the  bocly  the 
organisms  show  a  marked  preference  for  certain  special 
localities,  such  as  the  urethra,  the  meninges,  and  the  nasal 
cavities.  The  diseases  produced  are  on  the  whole  chronic 
with  a  marked  tendency  to  relapse,  and,  except  in  the  case  of 
the  meningococcus  infections,  rarely  terminate  fatally. 

Micrococcus  eatarrhalis. — This  coccus  is  the  least 
important  member  of  the  group,  and  may  be  regarded  as  a 
more  or  less  normal  inhabitant  of  the  nose  and  throat.  In 
film  preparations  it  appears  as  a  fairly  large  rounded  diplo- 
coccus,  and  is  often  found  within  the  phagocytes.  It  grows 
feebly  on  agar  in  small  colonies  when  first  isolated,  but 
more  abundantly  in  sub-cultures.  Growth  is  best  obtained  on 
blood  serum  media.  No  acidification  of  the  carbohydrate 
media  is  produced. 

The  organism  is  often  associated  with  the  common  "  in- 
fluenzal" catarrh,  and  may  be  obtained  in  such  cases  in 
pure  culture.  It  may  also  be  isolated  from  the  urine  in 
some  cases  of  cystitis. 

Micrococcus  melitensis. — This  coccus  is  the  causative 
organism  of  Malta  fever,  and  may  be  isolated  from  the 
blood  or  the  urine  of  infected  persons.  The  diagnosis  may 
be  confirmed  by  agglutination  tests  performed  with  the 
serum  on  the  coccus  in  the  same  manner  as  for  the  Grun- 
baum-Widal  reaction  of  typhoid  fever.  The  disease  is  spread 
by  the  drinking  of  goat's  milk,  a  widely  used  beverage  on 
the  Mediterranean  coasts.  The  cocci  are  present  in  large 
numbers  in  the  milk,  and  the  sera  of  the  infected  animals 
give  positive  agglutination  tests.  Malta  fever  is  a  disease 
attended  with  practically  no  danger  to  life,  but  it  runs  an 
extremely  protracted  course,  and  it  may  be  one  or  more  years 
before  the  patient  is  able  to  again  follow  his  ordinary  occu- 
pation. Since  the  discovery  of  the  causative  organism  and 
the  path  of  infection,  the  disease,  formerly  an  endless  source  of 
danger  to  the  British  troops  in  Malta,  has  been  almost 
eliminated. 

The  coccus  grows  very  poorly,  if  at  all,  at  20°  C,  and  at  body 
temperature  grows  slowly  on  agar,  the  small  colonies  taking 
about  three  days  to  reach  maturity.  Litmus  milk  is  rendered 
slightly  more  alkaline.  The  coccus  in  hanging-drop  preparations 


THE   COCCI— THE    GRAM-POSITIVE    BACILLI.     121 

is  definitely  motile,  and  has  been  described  as  flagellated 
by  some  observers.  In  films  made  from  the  media  it  appears 
in  pairs  and  small  clumps. 

The  meningococcus  (Diplococcus  intracellularis  menin- 
gitidis) (Plate  X.). — The  meningococcus  in  films  of  pus 
obtained  from  thecerebro-spinal  fluid  appears  as  a  diplococcus; 
some  of  the  organisms  are  extra-cellular,  but  the  majority  are 
within  the  phagocytes.  The  cocci  are  usually  rounded,  but 
occasional  pairs  with  flattened  opposed  surfaces  may  be  seen, 
and  rarely  a  polynuclear  cell  may  be  found  distended  with 
such  pairs,  the  organisms  thus  closely  resembling  the 
gonococcus  in  appearance.  Fortunately  it  is  practically 
never  required  to  distinguish  between  the  two  organisms, 
owing  to  the  diverse  situations  in  which  they  are  found. 
Meningococci  and  gonococci,  however,  are  readily  differentiated 
by  their  cultural  characters.  The  meningococcus  will  grow  on 
the  ordinary  media;  but  the  first  culture  is  usually  obtained 
with  difficulty,  and  frequent  sub-cultures  at  one-day  intervals 
are  necessary  before  a  ready  growth  results  on  agar  or  in 
broth.  It  is  advisable  to  make  the  first  cultures  on  to  nasgar 
or  some  other  blood  serum  medium  and  into  milk.  The  coccus 
grows  in  pin-point  colonies  on  nasgar  in  24  hours,  and  as  a 
rule  grows  freely  in  milk  without  changing  the  appearance  of 
the  medium.  The  coccus  is  usually  agglutinated  by  the 
serum  of  meningitis  cases  up  to  dilutions  of  1  in  50  or  over. 
The  meningococcus  is  the  causative  organism  of  cerebro-spinal 
meningitis,  both  in  its  epidemic  and  sporadic  form,  and  can 
nearly  always  be  obtained  in  pure  culture  from  the  cerebro- 
spinal fluid  during  life.  The  organism  has  also  been  found  in  the 
nose  and  throat,  and  is  probably  spread  from  these  situations. 
The  isolation  of  the  organism  from  a  nasal  discharge  is  a 
much  more  difficult  matter,  owdng  to  the  prevalence  of  other 
Gram-negative  cocci  of  the  catarrhalis  type.  The  organism 
cannot  be  identified  in  film  preparations,  and  the  chief  cultural 
points  of  distinction  are  the  failure  of  the  meningococcus  to 
grow  at  20°  C.  its  more  delicate  colonies  on  nasgar  and  its 
behaviour  in  the  carbohydrate  media.  Serum  agglutination 
tests  should  also  be  performed. 

The  gonococcus  (Plate  VIII.). — The  gonococcus  is 
characteristically  a  diplococcus  in  which  the  individual 
members  of  each  pair  have  the  opposed  surfaces  flattened. 


122  CLINICAL   PATHOLOGY. 

In  a  film  of  pus  the  great  majority  of  the  cocci  are  found 
within  the  phagocytes,  and  a  typical  field  shows  large  numbers 
of  empty  polynuclear  cells,  together  with  one  or  two  only  of 
the  polynuclear  or  epithelial  cells  crowded  with  diplococci.  A 
film  of  pus  in  which  the  comparatively  few  cells  which  are 
phagocytic  are  distended  with  flattened  Gram-negative  diplo- 
cocci, particularly  if  obtained  from  a  urethral  or  cervical 
discharge,  is  sufficiently  characteristic  to  practised  eyes  to 
ensure  the  diagnosis  of  a  gonorrheal  infection.  It  must  be 
remembered,  however,  that  errors  in  the  technique  of  using 
Gram's  stain  are  common,  and  that  an  intracellular  diplo- 
coccus  is  by  no  means  necessarily  a  gonococcus.  It  is 
unfortunate  that,  owing  to  the  difficulty  of  isolating  the 
gonococcus  in  culture,  the  diagnosis  has  often  to  be  made 
from  film  preparations,  and  in  cases  of  any  doubt  the  beginner 
should  be  far  more  cautious  than  is  customary  in  expressing 
an  opinion. 

The  gonococcus  will  not  grow  at  all  on  the  ordinary  media, 
but  requires  some  medium  containing  the  essential  elements 
of  blood  serum,  such  as  nasgar  or  serum  agar.  Perhaps  the 
best  medium  upon  which  to  grow  the  organism  is  an  agar 
slope  over  which  has  recently  been  smeared  with  a  platinum 
wire  a  drop  of  sterile  blood  obtained  by  pricking  the  thumb. 
On  this  medium  the  gonococcus  grows  well,  but  somewhat 
slowly,  the  small  "  pin-point  "  colonies  taking  from  24  to  48 
hours  to  reach  maturity.  Owing  to  the  slow  growth  of  the 
organism  it  is  likely  to  be  crowded  out  by  any  other  bacteria 
that  may  be  present  in  the  pus.  The  gonococcus  rapidly  dies 
out  in  culture  tubes  unless  frequent  subcultures  are  made. 

The  most  common  infections  produced  by  the  gonococcus 
are  intragenital — that  is  to  say,  a  urethritis  in  the  male  and  a 
urethritis,  or  more  commonly  an  inflammation  of  the  vagina 
and  cervix  uteri,  in  the  female.  The  diagnosis  of  the  nature 
of  an  acute  urethritis  in  the  male  is  easy ;  the  diagnosis  of 
gonorrhoea  in  the  adult  female  requires  a  special  examination. 
A  swab  taken  at  random  in  the  usual  manner  from  the 
vaginal  secretion  is  quite  useless.  Films  made  in  this  way,  even 
from  the  normal  vagina,  are  found  to  be  inundated  with  colon 
bacilli,  cocci,  and  other  organisms,  so  that  it  is  a  profitless  labour 
to  search  through  them  for  occasional  gonococci.  The  films 
should  be  made  from  the  interior  of  the  urethra  or,  after  the 


THE   COCCI— THE    GRAM-POSITIVE   BACILLI.     123 

passage  of  a  speculum,  from  the  cervical  canal,  care  being 
taken  not  to  touch  the  walls  of  the  vagina.  The  gonococcus  is 
the  commonest  cause  of  pyosalpinx,  but  can  very  rarely  be 
found  in  the  pus  obtained  at  operation.  Films  made  from  the 
pus  show,  as  a  rule,  numerous  phagocytes  but  no  cocci,  and 
cultures  prepared  from  it  remain  sterile.  The  infective 
vaginitis  of  little  girls  differs  in  some  clinical  respects  from 
the  gonorrhoea  of  adults,  in  that  it  is  extremely  contagious 
and  may  spread  through  a  children's  ward.  It  has  an 
unduly  long  incubation  period,  and  is  very  exceptionally 
followed  by  the  ordinary  sequelae,  such  as  arthritis.  Films 
made  from  the  vaginal  discharge  of  these  cases  are  found  to 
be  swarming  with  organisms  indistinguishable  from  gonococci. 

Among  the  complications  of  gonorrhoea  in  which  the  gono- 
coccus may  be  detected  are  conjunctivitis  (including  the  most 
frequent  variety  of  ophthalmia  neonatorum),  arthritis,  and 
rarely  a  general  septicaemia,  with  or  without  an  infective 
endocarditis. 

Commonly  included  among  the  cocci  must  be  mentioned 
a  group  of  organisms  known  as  Sarcinae  (Plate  IX.).  These 
are  non-pathogenic,  and  are  met  with  most  frequently  as  con- 
taminations in  culture  tubes  from  the  skin  or  from  the  air. 
In  film  preparations  they  appear  in  small  irregular  clumps,  or 
more  characteristically  in  little  packets  of  2,  4,  or  8,  the 
individual  members  dividing  at  right  angles  to  each  other. 
Their  opposed  surfaces  may  be  flattened.  The  colonies  on 
agar  are  usually  large,  round  and  opaque,  with  a  polished  and 
often  pigmented  surface.  Another  common  contamination  of 
culture  tubes  may  be  noticed  here,  namely,  the  bacillus 
subtilis,  which  can  be  recognised  by  its  usual  tendency  to 
abundant  spore  formation  and  the  pellicle  produced  by  it  on  the 
surface  of  broth  media. 

The  Gram-positive  Bacilli. 

These  may  be  divided  into  those  which  are  acid-fast  and 
those  which  are  not  acid-fast. 

(1)  The  acid-fast  bacilli.— This  important  group  of 
organisms  includes  a  considerable  variety  of  bacilli  some  of 
which  are  mainly  saprophytes ;  others  are  pathogenic  to 
animals  and  not  to   man  ;  while  the  few  more    particularly 


124  CLINICAL   PATHOLOGY. 

considered  here  are  highly  pathogenic  to  human  beings.  The 
organisms  resemble  each  other  in  that  they  do  not  grow  on 
the  ordinary  media,  and  when  growing  on  special  media  their 
rate  of  growth  is  extremely  slow.  The  lesions  produced  by 
them  resemble  each  other  in  their  nodular  or  tubercular 
characters  and  in  their  chronicity.  Although  the  bacilli  are 
all  acid-fast,  they  can  be  further  divided  by  their  staining 
reactions,  since  some  are  more  acid-fast  than  others,  and  some, 
though  acid-fast,  are  decolorised  by  alcohol,  that  is  to 
say,  are  not  alcohol-fast.  The  bacilli  have  a  tendency  to 
produce  long  thread-like,  beaded  forms,  particularly  in 
cultures,  and  are  no  doubt  fairly  closely  related  to  the 
streptothrices,  some  of  which  are  acid-fast  (page  145). 

The  tubercle  bacilli  (Plate  VIII.).— The  tubercle  bacilli 
can  be  recognised  in  films  made  from  human  lesions  and 
stained  by  the  Ziehl-Neelsen  method  (page  156).  They  appear 
as  thin,  curved  and  beaded  red  rods,  and  are  almost  entirely 
extracellular.  In  preparations  made  from  the  ordinary 
sources  such  bacilli  may  be  regarded  for  clinical  purposes  as 
tubercle  bacilli,  but  it  must  be  remembered  that  an  acid-fast 
bacillus  is  not  necessarily  the  human  tubercle  bacillus,  even  if 
found  in  human  tissues.  The  different  species  of  tubercle 
bacilli  can  be  differentiated  by  their  behaviour  in  culture  and 
by  animal  inoculation.  These  bacilli  may  be  divided  into 
those  which  are  pathogenic,  or  possibly  pathogenic,  to  man 
and  animals,  and  those  which  are  not.  The  pathogenic  bacilli 
include  the  following  : — 

The  human  tubercle  bacillus  is  by  far  the  most 
important  of  this  group  of  organisms,  and  is  responsible  for 
the  great  majority  of  all  human  tuberculous  lesions,  including 
nearly  all  cases  of  pulmonary  tuberculosis.  The  human  tubercle 
bacillus  will  grow  on  blood  serum,  or  on  media  to  which 
glycerin  has  been  added,  such  as  glycerin  agar  or  glycerin 
broth,  as  well  as  on  inspissated  egg  medium.  Growth  is  very 
slow,  and  little  is  to  be  seen  in  less  than  10  days  ;  later  it 
becomes  wrinkled,  greyish  and  scaly.  Inoculated  into 
animals  the  bacillus  is  found  to  be  highly  virulent  to  guinea- 
pigs  but  to  have  comparatively  little  virulence  for  rabbits  and 
calves. 

The  bovine  tubercle  bacillus  is  responsible  for  a 
proportion     of    the     glandular     and    arthritic     tuberculous 


THE   COCCI— THE   GRAM-POSITIVE   BACILLI.     125 

affections,  particularly  in  children,  and  much  less  frequently 
for  pulmonary  tuberculosis.  Owing  to  the  extreme  frequency 
of  tuberculosis  among  cows  and  the  common  presence  of 
bovine  bacilli  in  milk,  this  source  of  infection  for  the  human 
subject  is  no  doubt  a  wide  one  ;  at  the  same  time  the  typical 
human  infection  is  pulmonary  tuberculosis,  and  the  spread  of 
tuberculosis  in  its  most  important  aspect  is  by  the  human 
sputum.  The  bovine  bacillus  on  serum  grows  more  slowly 
than  the  human  bacillus,  taking  from  2  to  3  weeks  to  produce 
a  thin  greyish  film,  which  is  neither  wrinkled  nor  pigmented. 
It  is  highly  pathogenic  to  calves  and  rabbits. 

The  avian  tubercle  bacillus  appears  to  be  practically 
non-pathogenic  to  man,  and  since  tuberculosis  is  extremely 
rare  among  birds  in  the  wild  state,  though  common  among 
those  kept  in  captivity,  the  bacillus  can  scarcely  be  regarded 
as  a  serious  factor  in  the  spread  of  human  tuberculosis.  The 
avian  bacillus  is  readily  recognised  by  inoculation  experi- 
ments, since  it  is  highly  virulent  to  pigeons,  but  in  guinea-pigs 
produces  only  a  local  lesion. 

The  bacillus  of  fish  tuberculosis  does  not  grow  at  body 
temperature,  and  is  non-pathogenic  to  mammals. 

The  non-pathogenic  tubercle  bacilli  embrace  a  number 
of  acid-fast  organisms,  which  differ  from  the  pathogenic  bacilli 
in  their  far  more  rapid  growth  on  artificial  media.  They  are 
practically  non-pathogenic  to  animals,  and  on  inoculation  into 
the  highly  susceptible  guinea-pig  give  rise  to  a  small  local 
lesion  only.  These  bacilli  include  Rabinowitch's  butter 
bacillus,  Moeller's  timothy-grass  bacillus,  and  a  variety  of 
similar  organisms  found  in  dust,  manure,  and  other  substances. 
These  bacilli  rarely  if  ever  cause  confusion  in  human 
pathology. 

The  smegma  bacillus. — This  organism  belongs  properly 
to  the  group  of  non-pathogenic  tubercle  bacilli,  but  is 
considered  separately  because  of  its  human  distribution  and 
the  liability  to  confusion  with  the  virulent  tubercle  bacillus. 
The  smegma  bacillus  is  found  in  the  genital  secretions,  and  in 
film  preparations  appears  as  a  shorter,  stouter,  and  less  beaded 
rod  than  the  tubercle  bacillus.  The  smegma  bacillus  can  be 
readily  differentiated  by  its  staining  reactions,  since  after 
treatment  with  25  per  cent,  acid  it  is  decolorised  by  immer- 
sion  in   methylated  spirit  for  1  minute.      It  is,    therefore, 


126  CLINICAL   PATHOLOGY. 

acid-fast  but  not  alcohol-fast.  The  smegma  bacillus  has  been 
grown  with  difficulty  on  serum  and  glycerin- agar  media. 

For  the  purposes  of  clinical  pathology,  the  demonstration  of 
bacilli  which  are  strongly  acid-  and  alcohol-  fast  in  the  human 
tissues  is  sufficient  for  the  diagnosis  of  a  tuberculous  lesion. 
The  only  organism  likely  to  be  confounded  with  the  human 
tubercle  bacillus  is  the  bovine  bacillus,  and  for  practical 
diagnosis  the  two  may  be  considered  as  identical. 

In  tuberculosis  of  the  lung  and  of  the  urinary  tract  the 
bacilli  are  commonly  found  in  large  numbers  in  the  sputum 
and  urine.  In  the  pus  obtained  from  tuberculous  sinuses  and 
abscesses  or  from  tuberculous  joints  the  bacilli  are  almost 
always  extremely  scanty.  The  same  is  true  of  tuberculous 
body  fluids,  whether  peritoneal,  pleural,  or  cerebro-spinal ; 
also  of  the  skin  tuberculides.  In  the  faeces  the  bacilli  may  be 
present  in  large  numbers  in  cases  of  tuberculous  enteritis  and 
in  very  small  numbers  in  pulmonary  tuberculosis.  In  all 
situations,  other  than  the  urine  or  the  sputum,  it  is  necessary  to 
adopt  special  processes  for  the  demonstration  of  the  organisms, 
and  these  will  be  described  in  a  subsequent  chapter. 

The  lepra  bacillus.  —  The  leprosy  bacillus  closely 
resembles  in  appearance  the  human  tubercle  bacillus,  but  the 
following  distinctions  can  be  made  out.  The  bacilli  are  very 
numerous  in  any  leprous  lesion,  whether  in  the  pus  from  a 
breaking-down  focus,  or  from  a  sinus,  or  in  microscopic 
sections  of  lepra  nodules.  Many  of  the  bacilli  are  seen  within 
the  cells,  and  certain  elongated  cells  are  found  containing 
a  number  of  bacilli  arranged  like  a  bundle  of  cheroots. 
Further,  the  lepra  bacillus  is  not  so  acid-fast  as  the  tubercle 
bacillus,  but  is  decolorised  by  25  per  cent,  sulphuric  or  nitric 
acids,  retaining  the  stain  only  when  treated  with  acids  of  half 
this  strength.  The  lepra  bacillus  does  not  grow  on  the  media 
usually  employed  for  the  tubercle  bacillus,  and  until  recently 
had  never  been  cultivated. 

Cases  of  leprosy  are  extremely  rare  in  this  country,  and  all 
are  imported.  The  diagnosis  can  in  nearly  every  case  be 
confirmed  by  making  film  preparations  of  the  nasal  secretion. 
The  majority  of  leprous  individuals  have  a  chronic  crusted 
nasal  discharge  containing  the  bacilli  in  large  numbers,  so 
that  it  is  rarely  necessary  to  excise  a  suspected  nodule  for 
microscopic  examination. 


THE   COCCI— THE    GRAM-POSITIVE   BACILLI.     127 

In  spite  of  the  prevalence  of  the  bacilli  in  the  nasal  secretion 
the  disease  appears  to  have  very  little  direct  infectivity,  and  it 
is  held  by  certain  authorities  that  some  intermediate  host, 
such  as  a  biting  insect,  is  necessary  for  the  spread  of  the 
disease. 

(2)  The  non-acid-fast  bacilli. — This  small  group  of  Gram- 
positive  non-acid-fast  bacilli  consists  of  four  members  only, 
one  of  which  is  practically  non-pathogenic  to  man.  Each 
member  of  the  group  possesses  some  important  property  not 
usual  among  human  parasites ;  thus  two  members  produce 
powerful  extra  cellular  toxins,  two  are  strict  anserobes,  and  two 
form  spores.  One  member  of  the  group  only,  the  diphtheria 
bacillus,  has  any  wide  distribution  in  human  pathology. 

The  diphtheria  bacillus  (the  Klebs-Loffler  bacillus) 
(Plate  VIII.). — The  appearance  of  the  diphtheria  bacillus  in 
films  prepared  from  the  tissues,  and  more  particularly  from 
cultures,  is  very  characteristic.  The  bacilli  are  arranged  in 
small  groups,  and  the  members  of  each  group  have  a  peculiar 
angular  relation  to  each  other,  so  that  the  groups  resemble 
the  fingers  of  the  hands  crossed  or  a  Chinese  character.  The 
individual  bacilli  are  thin,  curved  and  beaded,  staining 
alternately  in  light  and  dark  areas.  They  are  non-motile. 
Some  bacilli  may  show  bulbous  or  racquet-shaped  extremities, 
and  these  so-called  "  involution  "  forms  are  common  in  cultures 
more  than  48  hours  old.  The  bacilli  are  readily  identified  from 
their  size,  shape,  beading  and  arrangement,  after  staining  with 
Loffler's  methylene  blue.  The  bacilli  grow  well  on  the 
ordinary  media,  but  it  is  preferable  to  make  the  first  culture 
on  ox  serum,  since  the  diphtheria  bacillus  grows  more  readily 
and  rapidly  on  this  than  the  ordinary  bacteria  present  in  the 
throat.  On  solid  media  the  bacilli  grow  in  colonies  of  the 
streptococcal  type,  but  they  are  slightly  more  heaped  up  and 
opaque.  A  general  turbidity  is  produced  in  broth,  and  if  a 
broth  culture  which  has  been  incubated  for  several  weeks  is 
filtered  free  from  bacilli  the  filtrate  contains  in  large  amount 
a  powerful  toxin,  which,  unlike  the  majority  of  bacterial 
toxins,  is  extracellular,  or,  in  other  words,  is  not  intimately 
bound  up  with  the  bodies  of  the  bacilli.  An  acid  reaction  is 
produced  within  24  hours  in  litmus  dextrose  broth,  and  the 
acidity  passes  off  again  in  a  few  days.  Gelatin  is  not  liquefied. 
If  a  suspension  of   the  bacilli  is  introduced  into  the  leg  of 


128  CLINICAL   PATHOLOGY. 

a  guinea-pig  death  results  in  about  36  hours,  and  iiost 
mortem  a  small  greyish  necrotic  membrane  is  found  at  the 
seat  of  inoculation.  The  stomach  is  greatly  dilated. 
Haemorrhages  are  present  in  the  supra-renals,  and  the 
cardiac  muscle  on  microscopical  examination  is  found  very 
extensively  affected  by  a  fine  fatty  degeneration.  If  a  second 
guinea-pig  is  inoculated  with  the  bacilli  or  their  toxins 
together  with  anti-diphtheritic  serum  no  ill  effects  result. 

The  diphtheria  bacillus  is  the  pathogenic  type  of  a  large 
number  of  bacilli,  the  non-pathogenic  members  being  known 
as  "diphtheroid"  bacilli. 

The  diphtheroid  bacilli  are  very  widely  distributed  in  the 
human  body,  and  it  is  essential  to  have  some  knowledge  of 
them  owing  to  the  extremely  close  resemblance  they  bear 
to  the  diphtheria  bacillus  itself.  The  diphtheroid  bacilli 
may  be  divided  into  those  which  can  be  distinguished  from 
the  Klebs-Lofner  bacillus  on  morphological  grounds,  those 
which  cannot  be  so  distinguished  but  which  have  cultural 
differences,  and  those  which  are  both  morphologically  and 
culturally  identical.  Hofmann's  bacillus  can  be  distinguished 
on  morphological  grounds  by  its  appearance  in  film  prepara- 
tions made  from  a  culture.  The  bacilli  are  distributed  in 
groups,  the  members  of  which  have  a  tendency  to  a  parallel 
rather  than  an  angular  arrangement.  They  are  short  and 
rarely  curved,  and  are  not  truly  beaded,  but  stain  deeply 
at  each  end,  displaying  a  pale  band  across  the  middle.  There 
is  some  doubt  as  to  the  ability  of  the  Hofmann  bacillus  to 
produce  disease  or  as  to  whether  it  is  capable  of  conversion 
into  the  diphtheria  bacillus,  and  it  is  wiser  to  treat  patients 
from  whom  this  bacillus  is  isolated  as  infective,  particularly  if 
a  lesion  in  any  way  suggestive  of  diphtheria  is  present. 

Bacilli  are  commonly  met  with  in  the  conjunctival  sac  (in 
which  situation  they  have  undeservedly  obtained  the  name  of 
Xerosis  bacilli),  in  the  skin,  in  old  sinuses,  and  less  frequently 
in  the  nose,  which  are  morphologically  identical  with  the 
diphtheria  bacillus,  differing  from  it  only  in  a  greater  tendency 
to  produce  club-shaped  forms.  The  niajorhVv  of  these  bacilli 
can  be  differentiated  on  cultural  grounds,  since  they  merely 
maintain  an  existence  in  liquid  media  and  produce  little  or  no 
growth  in  them.     They  do  not  acidify  dextrose  broth. 

Other  bacilli,  which  are  frequently  isolated  from  the  urethra 


THE   COCCI— THE    GRAM-POSITIVE    BACILLI.     129 

and  less  often  from  any  of  the  above  situations,  resemble  the 
diphtheria  bacillus  exactly  both  in  their  morphology  and  in 
their  cultural  characters.  They  differ,  however,  in  one  very 
important  respect — they  produce  no  toxin,  and  are  consequently 
non-pathogenic  to  guinea-pigs. 

In  practical  pathology,  if  a  bacillus  is  obtained  from  the 
throat  or  larynx  of  a  person  whose  clinical  condition  suggests 
diphtheria,  and  if  it  is  found  to  resemble  the  diphtheria  bacillus 
in  appearance,  the  patient  should  be  isolated  and  given 
antitoxin.  In  the  case  of  nasal  diphtheria  the  full  cultural  and 
morphological  characters  of  the  bacilli  should  be  ascertained, 
since  diphtheroid  bacilli  are  common  in  this  situation,  and 
greater  attention  should  be  paid  to  the  clinical  condition  than 
to  the  bacteriological  findings.  In  patients  convalescent  from 
diphtheria  it  is  more  practical  to  let  them  mix  again  with 
their  fellows,  if  they  are  free  from  tonsillar  or  nasal  discharge, 
than  to  confine  them  on  purely  bacteriological  grounds.  In  sus- 
pected diphtheritic  lesions  in  unusual  places,  such  as  the  con- 
junctiva, surface  wounds,  the  vagina,  etc.,  the  morphological 
and  cultural  characters  of  the  bacillus  are  insufficient  for  diag- 
nosis. In  all  cases  of  doubt  or  of  exceptional  importance  (such 
as  a  sporadic  case  of  diphtheria  in  a  school)  the  bacilli  must 
be  isolated  and  animal  inoculations  performed. 

The  routine  bacteriological  examination  of  the  throat  is 
therefore  of  great  assistance  in  suspected  cases  of  diphtheria, 
but  the  results  should  be  accepted  with  reserve  in  other 
situations,  and  should  not  be  regarded  as  proven  until  a 
guinea-pig  has  been  inoculated. 

The  successful  isolation  of  the  bacilli  from  the  throat  is 
largely  dependent  upon  the  care  with  which  the  culture  is 
taken.  The  necessary  apparatus  consists  of  a  blood  serum 
culture  tube  and  a  second  tube  containing  a  straight  piece  of 
stout  copper  wire,  around  one  end  of  which  has  been  firmly 
twisted  a  piece  of  absorbent  wool.  Wool  and  wire  must  be 
sterile.  In  the  case  of  a  refractory  child  have  the  arms,  legs, 
and  body  wound  in  a  blanket  and  the  child's  head  held  by  a 
nurse.  Choose  a  good  light,  depress  the  tongue  with  a  spatula, 
pass  the  cotton  wool  swab  on  to  the  membrane  and  rub  it 
firmly  into  the  lesion.  Withdraw  without  touching  the  tongue 
or  cheeks.  Rub  the  swab  over  the  surface  of  the  serum  and 
replace  in   its   own    tube   without   burning  it.     Incubate  for 

p.  9 


130  CLINICAL  PATHOLOGY. 

12  hours.     Examine  films  stained  with  Loffler's  methylene 
blue  from  both  swab  and  medium. 

The  acne  bacillus  is  an  organism  of  low  pathogenicity 
belonging  to  this  group.  In  film  preparations  it  resembles 
the  diphtheria  bacillus  in  its  grouping,  but  is  smaller  and 
is  not  beaded,  being  in  appearance  not  unlike  Hofmann's 
bacillus.  It  grows  with  difficulty  on  the  ordinary  media 
except  in  sub-culture,  and  the  first  culture  is  best  planted 
on  oleic  acid  glycerin  agar,  and  grown  under  anaerobic 
conditions.  The  acne  bacillus  is  obtained  in  consider- 
able numbers  from  the  depths  of  acne  comedones.  When 
suppuration  occurs  a  white  staphylococcus  is  usually  present 
in  addition. 

The   tetanus    bacillus    (Plate    VIII.).— Tetanus   bacilli 
when  growing  in  the  tissues  commonly  have  few,  if  any,  spore - 
bearing  forms.     On  artificial  media  the  majority  of  the  bacilli 
contain    spores.     The    bacillus    under    these    conditions    is 
comparatively   slender,   and   the   delicate   rounded   spore    is 
situated  at  one  extremity.     Other  spores  are  seen  lying  free 
among   the   bacilli.     The    bacillus    is    feebly    motile    and    is 
provided  with  flagella.     The  tetanus  bacillus  usually  maintains 
its  existence  at   the   depths   of   septic   wounds,  and  for  this 
reason    is    difficult    to    identify    and    isolate.      One   method 
of  isolation   presumes    the    existence    of    spores   which   are 
very  resistant  to  heat,  and  on  this   supposition  a  series  of 
melted  agar  tubes  are   inoculated   at   varying  temperatures 
in  the  hope  that  other  organisms  will  be  destroyed  and  the 
tetanus  spores  survive.      The  culture  tubes  must  be  grown 
under  strictly  anaerobic  conditions.     In  agar  and  gelatin  stab 
cultures  the  bacillus  sends  radiating  spikes  into  the  media, 
and  the  gelatin  is  slowly  liquefied.      Such  cultivation  of  a 
bacillus  with  terminal  spores  is  extremely  suggestive  of  the 
tetanus   organism;    the   positive   proof,   however,  rests  with 
animal  inoculation.     Subcutaneous  inoculation  of  the  bacilli 
into  a  mouse  produces  tetanic  spasms  in  24  hours  and  death 
m  3  days.     The  tetanus  toxin  is  an  extremely  powerful  one, 
and,  like  the  diphtheria  toxin,  is  extracellular. 

The  anthrax  bacillus  (Plate  VIII.).— Anthrax  infections 
in  man  are  extremely  rare  and  occur  in  two  main  forms.  The 
more  usual  variety  is  a  local  infection  of  the  skin,  arising  among 
hide  porters  and  workers,  and  known  as  the  malignant  pustule. 


THE   COCCI— THE    GKAM-POSITIVE   BACILLI.     181 

Recovery  from  this  form  under  treatment  is  usual.  The  other 
variety  is  known  as  wool-sorters'  disease,  and  in  this  the  local 
infection  is  in  a  bronchus,  extension  taking  place  through  the 
bronchial  glands.  This  form  is  invariably  fatal.  In  films 
made  from  the  clear  fluid  of  the  bleb  of  a  malignant  pustule 
occasional  polynuclear  cells  are  found,  and  large  numbers  of 
long,  stout  bacilli,  with  sharply  cut  ends,  are  seen.  The 
majority  of  the  bacilli  are  arranged  in  long  chains,  each  chain 
being  contained  within  a  delicate  capsule.  In  films  made  from 
old  cultures  the  great  majority  of  the  bacilli  are  seen  to  contain 
central  spores  ;  not  infrequently  spores  only  are  found  and  no 
bacilli.  The  bacillus  grows  readily  on  all  the  ordinary  culture 
media,  the  most  characteristic  growths  being  found  on  agar 
plates  and  in  gelatin  stabs.  On  agar  plates  the  colonies 
examined  with  a  magnifying  glass  appear  like  wreathed  coils 
of  hair,  an  appearance  produced  by  the  long-twisted  strands  of 
bacilli.  In  gelatin  stab  cultures  growth  occurs  along  the  line 
of  the  stab,  and  branches  out  from  this  in  spikes  radiating 
into  the  medium.  The  spikes  nearest  the  surface  are  the 
longest,  so  that  the  growth  looks  like  an  inverted  fir  tree. 
Liquefaction  of  the  gelatin  begins  at  the  surface  on  about  the 
second  day  of  the  growth. 

The  bacillus  aerogenes  capsulatus. — This  bacillus  is  of 
little  pathological  importance,  but  may  give  rise,  usually  in 
association  with  other  organisms,  to  emphysematous  gangrene 
and  to  gas-containing  abscesses.  It  is  more  commonly  found 
post  mortem  as  a  gas-producing  putrefactive  organism.  The 
bacillus  is  a  strict  anaerobe  growing  in  capsulated  chains  on 
the  ordinary  media.  It  produces  abundant  gas  in  the 
carbohydrate  media,  and  does  not  appreciably  liquefy  gelatin. 


9—2 


CHAPTEE  X. 

THE    GEAM-NEGATIVE    BACILLI — SPIRILLA — STREPTOTRICHEjE — 

hyphomycetes. 
The  Gram-negative  Bacilli. 

The  great  majority  of  bacilli  being  Gram-negative,  it  is 
convenient  to  further  subdivide  them  into  a  number  of  groups, 
mainly  according  to  their  similarity  in  cultural  characters. 

Group  1. 

This  group  consists  of  four  members,  two  of  which  produce 
acute  specific  fevers;  two  are  common  causes  of  eye  affections. 
There  is  great  morphological  and  cultural  similarity  between 
the  members. 

The  whooping-cough  bacillus  (the  Bordet-Gengou 
bacillus),. — A  great  variety  of  organisms  have  been  described 
as  the  essential  cause  of  pertussis.  The  bacillus  referred  to 
here  is  that  isolated  by  Bordet  and  Gengou  and  proved  by 
them  to  be  the  causative  organism.  The  bacillus,  which  is 
found  in  considerable  numbers  in  the  sputum  in  the  early 
stages  of  whooping-cough,  is  a  minute  one  closely  resembling 
the  influenza  bacillus,  but  in  culture  has  less  tendency  to 
produce  involution  forms,  and  on  subculture  grows  more 
abundantly.  The  bacillus  is  agglutinated  by  the  sera  of 
patients  convalescent  from  whooping-cough,  and  its  specificity 
has  been  further  demonstrated  by  complement  fixation 
tests. 

The  influenza  bacillus  (Pfeiffer's  bacillus)  (Plate  VIII.). 
— In  films  made  from  the  sputum  the  bacilli  appear  as  tiny 
rods,  often  in  clumps  of  considerable  size,  and  many  of 
them  are  found  within  the  phagocytes.  The  organisms  are 
present  also  in  many  of  the  complications  of  influenza,  and 
may  be  obtained  in  pure  culture  from  empyema  pus  and 
from  joint  fluids.  A  purulent  exudate  in  which  the  polynu- 
clear  cells  contain  minute  Gram -negative  bacilli,  and  in  addition 


PLATE  IX. 


B.  Pestis.  Cholera  Vibrio. 

In  Smear  from  Spleen.    (Carbol-thionin.)  Broth  Culture.    (Carbol-thionin.) 


B.  Coli.  Actinomyces. 

Broth  Culture.    (Carbol-thionin.)  Filaments  in  Sputum  (Gram's  Stain. 


Spiroehaeta  Pallida.*  Spirilla  and  Fusiform  Bacilli  (Vincent). 

Film  from  Chancre.    (Giemsa's  Stain.)  From  Septic  Mouth.     (Carbol-thionin.) 


Oidium  Albicans.  Sarcinse. 

From  Agar  Culture.     (Carbol-thionin.)  From  Agar  Culture.     (Carbol-thionin.) 

*  The  sharpness  and  regularity  of  the  spirals  are  to  a  large  extent  lost  in  the  process 
of  reproduction. 


PLATE    IX. 


'  )   ~ 

) 

i    ',  '. 

\ 

I     Li         »    - 

,N 

1     u 

J 

V 

■0>,V^    ^ 

)     ^             f 

' 

1     ' 

J 

v>-« 

V,    "*           „ 

0   j 

i 

S.  s 

'    e        > 

V 

-s 

'»        *'    ~ '  £  i 


...        'V  <" 


GRAM-NEGATIVE  BACILLI— SPIRILLA,   ETC.     133 

clumps  of  the  same  bacilli  lying  free,  is  an  exudate  caused  by 
one  of  the  bacilli  in  this  group.  In  cultures  the  influenza 
bacillus  will  grow  only  on  media  containing  serum,  and 
preferably  also  haemoglobin,  the  best  medium  being  an  agar 
slope  streaked  with  fresh  blood.  The  colonies  are  very  small 
and  very  translucent.  While  there  is  no  doubt  that  this 
bacillus,  described  first  by  Pfeiffer  and  Kitasato,  is  the  causative 
organism  of  some  influenzal  epidemics,  it  must  not  be  expected 
to  recognise  it  in  all  cases  of  so-called  influenza.  The 
diagnosis  of  influenza  is  often  a  haphazard  one,  and  in  some 
undoubted  epidemics  there  has  been  a  failure  to  discover 
Pfeiffer's  organism;  moreover,  there  is  possibly  a  variety  of 
different  organisms  associated  with  a  number  of  similar 
clinical  conditions. 

The  Koch- Weeks  bacillus. — This  bacillus  is  by  far  the 
most  common  cause  of  acute  contagious  conjunctivitis.  The 
organisms  are  almost  identical  in  appearance  with  the 
influenza  bacillus,  and  are  found  in  large  numbers  in  films 
made  from  the  conjunctival  discharge.  The  cultural  properties 
of  the  bacillus  also  closely  resemble  those  of  Pfeiffer's 
organism,  but  the  more  favourable  media  are  serum  or  ovarian 
agar. 

The  Morax-Axenfeld  bacillus. — This  bacillus  is  the 
cause  of  diplo-bacillary  or  angular  conjunctivitis,  a  common  but 
less  acute  form  of  contagious  conjunctivitis  than  the  preceding. 
The  bacillus  appears  as  a  fairly  large,  broad  bacillus  with 
rounded  ends,  mainly  in  pairs,  but  also  in  short  chains.  It  is 
found  in  the  conjunctival  secretion,  and  in  a  fair  proportion  of 
cases  in  the  nasal  discharge  also.  It  can  be  cultivated  on  blood 
serum  media,  and  grows  in  colonies  of  the  streptococcal  type. 

Group  2. 

This  small  group  consists  of  two  members  only,  which  differ 
in  many  respects  from  the  bacilli  of  the  preceding  group. 
Both  organisms  are  met  with  but  rarely  in  the  human 
pathology  of  this  country. 

B.  Mallei. — The  bacillus  of  glanders  mainly  affects  horses, 
and  very  exceptionally  produces  disease  in  man.  Infection  in 
man  starts  from  a  local  abrasion  on  the  skin  or  the  nasal 
mucous  membrane  and  spreads  by  the  lymphatics,  giving  rise 
to   an   acute   or    chronic  pysemic   condition,   with  secondary 


134  CLINICAL   PATHOLOGY. 

abscesses  in  the  tissues,  lungs,  or  joints.  The  bacilli  are 
slender,  curved  rods,  staining  faintly,  and  often  in  a  beaded 
manner,  with  the  ordinary  dyes.  In  film  preparations  they 
are  mainly  extracellular.  The  organism  grows  on  the 
ordinary  media,  but  somewhat  slowly.  On  agar  and  blood 
serum  growth  appears  as  a  shiny,  greyish  streak  in  two  days. 
On  rjotato  a  membranous  growth  is  formed,  which  by  the 
eighth  day  becomes  a  reddish  brown  colour.  Inoculation  into 
the  abdominal  cavity  of  a  male  guinea-pig  is  followed  by 
peritonitis,  swelling  of  the  testicles,  and  a  purulent  exudate 
into  the  tunica  vaginalis.  The  appearance  and  staining 
reactions  of  the  organism,  the  growth  on  potato,  and  the 
effect  of  inoculation  into  a  guinea-pig  should  all  be  investi- 
gated before  making  a  diagnosis. 

B.  Pestis  (Plate  IX.). — The  bacillus  of  plague  is  mainly 
spread  to  man  from  the  rat  by  the  intermediary  of  the  rat  flea. 
The  disease  in  man  is  of  three  main  types — the  bubonic,  in 
which  the  lymphatic  glands  are  affected,  the  pulmonary,  which 
almost  invariably  terminates  in  a  fatal  septicaemia,  and  the 
septicaemia.  The  bacilli  are  found  in  large  numbers  in  films 
made  from  gland  pus  or  sputum,  and  stain  deeply  at  each 
end  and  faintly  in  the  centre,  each  bacillus  having  the 
appearance  of  a  diplococcus.  This  polar  staining  is  best  seen 
if  the  films  are  first  fixed  in  absolute  alcohol.  B.  pestis  grows 
on  the  ordinary  media,  forming  a  streak  on  agar.  Gelatin  is 
not  liquefied.  Broth  shows  a  granular  deposit,  and  the  bacillus 
grows  in  chains.  The  disease  can  be  reproduced  in  mice  and 
guinea-pigs  by  inoculation. 

Group  3  a. 

The  third  group  of  Gram-negative  bacilli  comprises  a  con- 
siderable variety  of  organisms,  the  great  majority  of  which  are 
morphologically  indistinguishable  from  one  another.  The 
group  can  be  subdivided  on  the  basis  of  one  important 
cultural  characteristic,  namely,  the  manner  of  growth  on 
gelatin.  The  bacilli  of  the  first  division  include  all  the 
important  organisms  of  the  coli-typhoid  group,  and  do  not 
liquefy  gelatin.  The  bacilli  of  the  second  division  are  of  less 
pathological  significance,  and  all  liquefy  gelatin.  Group  3a 
contains  the  typhoid  and  paratyphoid  bacilli,  the  dysentery 
bacilli,  and  the  colon  bacilli. 


GRAM-NEGATIVE   BACILLI— SPIRILLA,   ETC.     135 

B.  Typhosus. — The  typhoid  bacillus  is  an  actively  motile 
organism  provided  with  numerous  flagella.  Rapid  growth  is 
readily  obtained  on  all  the  usual  cultural  media.  The  more 
important  cultural  characters  are  the  following.  On  agar 
slopes  a  streak  is  produced  which  is  rather  more  translucent 
and  has  less  tendency  to  lateral  spread  than  in  the  case  of  the 
colon  bacillus ;  the  differences,  however,  are  not  pronounced. 
In  broth  a  general  turbidity  is  produced,  but  no  indole.  In 
litmus  milk  a  faint  acid  reaction  is  set  up,  but  no  clotting 
takes  place.  No  gas  is  produced  in  any  of  the  litmus 
carbohydrate  media,  but  litmus  dextrose  is  acidified.  Neutral 
red  broth  is  unchanged.  Yellow  colonies  are  produced  on 
MacConkey's  neutral  red  bile-salt  lactose  agar  medium. 

The  bacillus  can  be  identified  by  its  appearance,  staining 
reactions  and  cultural  characters,  and  the  identification  is 
preferably  confirmed  by  making  use  of  the  agglutination  test 
with  a  known  typhoid  serum. 

Typhoid  fever  is  spread  either  by  direct  contact,  such  as 
may  occur  in  nursing  a  patient,  or  more  commonly  by  an 
infected  water  supply.  The  disease  is  in  the  first  instance  a 
general  blood  infection,  and  the  bacilli  may  be  readily  isolated 
from  the  blood  in  the  first  week  of  the  disease  and  during  a 
relapse.  Later  the  bacilli  are  found  in  the  faeces,  and  less 
commonly  in  the  urine.  The  bacilluria  which  may  complicate 
the  late  stages  of  typhoid  fever  is  usually  set  up  by  the  colon 
bacillus,  and  far  less  commonly  by  the  typhoid  bacillus.  The 
bacilli  may  be  obtained  from  the  gall  bladder  many  years 
after  the  attack,  and  are  present  in  the  bone  abscesses  which 
may  follow  the  attack.  The  bacilli  may  persist  in  the  fgeces, 
and  less  commonly  in  the  urine,  many  years  after  the  primary 
infection  and  without  injury  to  the  host.  Persons  thus 
infected  are  known  as  "  typhoid  carriers,"  and  are  a  grave 
source  of  danger  to  the  community.  The  laboratory  diagnosis 
of  typhoid  fever  can  be  made  in  the  first  week  of  the  disease 
by  means  of  a  blood  culture.  A  few  cubic  centimetres  of  blood 
are  taken  into  broth  or  into  ox  bile,  incubated  for  12  hours, 
and  plated  out  on  MacConkey's  medium.  The  yellow  colonies 
are  then  picked  off  and  tested  culturally  and  by  the  serum 
reaction.  At  the  beginning  of  the  second  week  the  Grunbaum- 
Widal  test  becomes  positive  in  the  patient's  serum  and  the, 
bacilli  may  be  isolated  from  the  feces. 


136  CLINICAL   PATHOLOGY. 

Prophylactic  inoculation  against  typhoid  fever  with  dead 
cultures  of  the  bacilli  is  advisably  given  to  those  compelled  to 
live  in  countries  where  the  disease  is  rife.  Persons  thus 
inoculated  should  not  abate  the  customary  precautions  taken 
with  regard  to  their  water  supply,  since  they  are  not  immune 
against  a  heavy  infection. 

The  paratyphoid  bacilli. — The  paratyphoid  bacilli  form 
a  small  group  of  closely-allied  organisms  which  can  be  recog- 
nised generally  by  their  cultural  characters,  specifically  only 
by  agglutination  tests.  Four  members  of  the  group  may  be 
mentioned  here  : — B.  paratyphosus  A  ;  B.  paratyphosus 
B  ;  B.  suipestifer  ;  B.  enteritidis  (Gaertner).  The  first  two 
bacilli  produce  a  disease  which  cannot  be  distinguished  on 
clinical  grounds  from  typhoid  fever.  Paratyphoid  infections, 
however,  commonly  run  a  milder  course  than  typhoid,  are 
perhaps  more  contagious,  are  to  be  suspected  when  patients 
apparently  suffering  from  typhoid  fail  to  give  positive  Widal 
reactions,  and  are  definitely  diagnosed  on  the  agglutination 
tests  with  the  appropriate  bacilli,  and  on  the  isolation  of  the 
organisms  from  the  blood  or  f&ces.  The  paratyphoid  B 
bacillus  is  the  organism  most  commonly  met  with  in  this 
country,  while  numerous  cases  of  infection  by  the  A  bacillus 
have  been  recorded  from  America  and  India.  B.  suipestifer 
and  B.  enteritidis  (Gaertner)  are  commonly  associated  with 
meat-poisoning  epidemics,  and  produce  a  disease  with  an 
incubation  period  of  a  few  hours,  followed  by  acute  gastro- 
intestinal symptoms  of  comparatively  short  duration. 

The  organisms  as  a  group  have  the  following  cultural 
characters.  Acid  and  gas  are  produced  in  litmus  dextrose  and 
litmus  mannite.  Litmus  lactose  is  unchanged.  No  indole 
is  formed.  Litmus  milk  becomes  acid  in  the  first  24  hours 
and  then  strongly  alkaline.  Neutral  red  broth  is  turned 
yellow.  B.  ■paratyphosus  A  differs  from  the  other  members  of 
the  group  in  its  behaviour  in  litmus  milk,  in  which  it  produces 
a  permanent  acidity  but  no  clot. 

The  bacilli  can  be  differentiated  one  from  the  other  by  care- 
fully performed  absorption  tests  with  the  sera  of  infected 
persons  on  the  lines  indicated  in  a  previous  chapter.  Such 
tests  are  rarely  required  in  clinical  pathology.  Suspected 
cases  of  typhoid  fever  which  give  in  their  sera  negative  or 
partial   agglutination   reactions   with  B.  typhosus  should  be 


GEAM-NEGATIVE   BACILLI— SPIKILLA,   ETC.     137 

tested  upon  a  known  culture  of  B.  paratyphosus  B.  The 
organism  should  also  be  sought  for  in  the  blood  and  in  the 
fseces,  and  by  its  milk  reaction  it  can  be  distinguished  cultur- 
ally from  the  A  bacillus.  Cases  of  acute  food  poisoning 
should  in  the  later  stages  of  infection  be  tested  for  agglutinins 
against  Gaertner's  bacillus  and  the  organism  sought  for  in  the 
blood  or  fseces  in  the  acute  stage,  as  well  as  in  the  suspected 
food. 

The  dysentery  bacilli. — The  dysentery  bacilli  form  a 
small  group  of  closely-allied  and  widely-spread  organisms,  of 
which  the  main  varieties  are  the  B.  dysentcrice  of  Shiga  and 
the  B.  dysenteries  of  Flexner.  These  organisms  are  the 
causes  of  bacillary  dysentery,  such  as  is  met  with  in  tropical 
and  in  temperate  climates.  They  are  also  associated  with 
some  forms  of  ulcerative  colitis  in  this  country,  and  have  been 
found  in  outbreaks  of  asylum  and  other  varieties  of  dysentery, 
as  well  as  in  epidemics  of  infantile  enteritis. 

The  bacilli  are  practically  non-motile,  and  do  not  appear  to 
be  provided  with  flagella.  Their  chief  cultural  characters  are 
as  follow  : — No  gas  is  produced  in  any  of  the  carbohydrate 
media.  Litmus  glucose  is  acidified,  and  litmus  lactose  is 
unchanged.  Litmus  mannite  is  acidified  by  the  Flexner 
organism,  but  not  by  the  Shiga  bacillus.  No  indole  is  formed. 
Milk  is  rendered  first  acid  and  then  alkaline.  Neutral  red 
broth  is  unchanged. 

In  suspected  cases  of  dysentery  the  bacilli  should  be  sought 
for  in  the  stools,  and  the  patient's  serum  should  be  tested  by 
ordinary  agglutination  methods  upon  the  bacilli  isolated  and 
upon  known  strains  of  dysentery  bacilli.  In  a  positive  serum 
reaction  the  bacilli  are  agglutinated  somewhat  slowly  in 
dilutions  of  the  serum  up  to  1  in  50. 

The  colon  bacilli  (Plate  IX.) — The  Bacillus  coll  communis, 
like  other  members  of  this  group,  has  certain  typical  characters, 
but  occasional  varieties  are  met  with  presenting  differences  of 
minor  importance.  The  typical  bacillus  only  will  be  con- 
sidered here. 

The  colon  bacillus  has  few  flagella,  and  is  only  sluggishly 
motile,  though  active  strains  are  occasionally  met  with.  The 
organism  can  be  identified  by  its  cultural  characters,  never  from 
its  morphological  appearances  alone.  The  main  cultural 
reactions  are  as  follow.     Acid  and  gas  are  produced  in  the 


138  CLINICAL   PATHOLOGY. 

great  majority  of  the  carbohydrate  media,  including  dextrose, 
lactose,  and  mannite.  Indole  is  produced  in  broth.  Milk  is 
acidified  and  clotted.  On  agar  slopes  a  thick  greyish  white 
streak  is  formed  with  spreading  edges  ;  on  agar  plates  large 
circular  colonies  appear  with  heaped-up  centres  and  crenated 
margins.  (The  appearances  on  solid  media  are  almost  iden- 
tical for  all  members  of  the  colon-typhoid  group,  except  that 
the  typhoid  growth  is  slightly  more  delicate,  while  the  other 
organisms  occupy  an  intermediate  position.)  The  colour  of 
neutral  red  broth  is  changed  to  a  canary-yellow,  and  a  green 
fluorescence  is  produced  in  the  medium.  Eed  colonies  are 
formed  on  MacConkey's  bile  salt  medium. 

The  colon  bacillus  is  a  normal  inhabitant  of  the  large 
intestine,  but  in  abnormal  situations  it  produces  suppuration 
and  disease.  The  more  important  affections  associated  with 
this  organism  are  those  connected  with  intestinal  lesions,  as, 
for  example,  the  general  peritonitis  which  follows  a  perforated 
appendix.  The  colon  bacillus  frequently  gains  entry  into  the 
urinary  tract,  particularly  of  females,  where  it  may  lie  latent, 
producing  no  symptoms,  or  may  give  rise  to  severe  suppura- 
tive nephritis,  pyelitis,  or  cystitis.  The  bacillus  is  also  found 
in  diseased  processes  in  the  gall  bladder  (often  in  association 
with  calculi),  in  the  bile  passages,  and  in  numerous  other  parts 
of  the  body.  It  has  even  been  isolated  in  pure  culture  from 
the  cerebro -spinal  canal.  It  is  rarely  isolated  from  the  general 
circulation,  except  shortly  before  death. 

The  pus  in  which  the  colon  bacillus  is  found  is  frequently 
most  offensive,  owing  to  the  fact  that  since  these  lesions  are 
so  often  in  communication  with  the  gastro-intestinal  tract 
there  are  present  in  addition  to  the  colon  bacilli  certain  long, 
thin,  delicate  saprophytic  bacilli  which  normally  inhabit  this 
tract,  and  are  capable  of  producing  the  most  virulent  odour. 
It  is  this  odour  to  which  the  surgeon  is  apt  to  call  attention 
as  typical  of  the  bacillus  coli,  which  may,  however,  be  absent. 

Serum  reactions  with  the  bacillus  are  unsatisfactory,  since 
an  appreciable  increase  in  agglutinin  is  not  commonly  present, 
and  the  sera  of  infected  persons  rarely  agglutinate  the  bacillus 
in  dilutions  of  more  than  1  in  10. 

Note. — The  student  is  scarcely  expected  to  remember  in 
detail  the  various  cultural  characters  of  the  organisms  in  this 
group  (Group  3a).     He  should,  however,  remember  the  main 


GRAM-NEGATIVE   BACILLI— SPIRILLA,   ETC.     139 

distinctions  between  the  typhoid  and  colon  bacilli.  A  reference 
to  the  bacteriological  table  affords  a  simple  guide  to  these 
characters.  The  bacilli  are  placed  from  above  downwards  in 
order  of  their  virulence,  their  specificity,  their  motility,  and 
their  properties  of  producing  agglutinins.  The  cultural 
characters  proceed  in  the  opposite  direction  ;  thus  the  colon 
bacillus  alters  almost  every  medium  in  the  greatest  possible 
manner,  the  typhoid  bacillus  has  the  least  effect  upon  the 
media,  and  the  other  organisms  occupy  an  approximately 
intermediate  position. 

Group  3b. 

The  members  of  this  group,  all  of  which  liquefy  gelatin,  are 
not  of  great  pathological  importance.  They  may  be  described 
as  bacilli  which  usually  lead  a  saprophytic  existence,  but  may 
on  occasion,  either  alone  or  more  commonly  in  association  with 
other  organisms,  produce  disease.  Only  three  members  of  the 
group  are  considered  here. 

B.  Proteus- — This  bacillus  is  represented  by  a  number  of 
closely-allied  organisms,  which  differ  somewhat  in  their 
cultural  characters.  The  bacilli  in  appearance  are  indis- 
tinguishable from  the  colon  bacillus,  and  are  very  sluggishly 
motile.  In  cultures  they  produce  acid  and  gas  in  several 
carbohydrate  media,  but  as  a  rule  do  not  change  lactose.  A 
yellow  colour  with  a  marked  green  fluorescence  is  rapidly  pro- 
duced in  neutral  red  broth.  Milk  is  unchanged  by  some 
members  of  the  group  and  acidified  by  others.  The  colonies 
on  MacConkey's  neutral  red  bile-salt  lactose  agar  are  yellow. 
Abundant  indole  is  produced  in  broth.  B.  proteus  is  commonly 
found  in  the  fseces  and  in  the  urine.  In  septic  infections  of 
the  urinary  tract  the  organism  may  be  recovered  in  pure 
culture,  or  more  commonly  in  association  with  the  colon 
bacillus.  The  presence  of  B.  proteus  in  a  carefully  taken 
catheter  specimen  of  urine  is  suggestive  of  some  underlying 
organic  lesion  such  as  tuberculosis,  malignant  disease,  or 
calculus,  since  a  primary  infection  with  this  organism,  unlike 
a  primary  infection  by  the  colon  bacillus,  is  most  unusual. 

B.  Pyocyaneus. — This  is  an  actively  motile  bacillus  with  a 
tendency  to  spontaneous  agglutination  in  hanging-drop  prepara- 
tions. The  organism  is  readily  recognised  in  cultures  by  the 
bright  green  pigment  which  it  produces.      Broth  is  turned  a 


140  CLINICAL   PATHOLOGY. 

vivid  green  (without  fluorescence),  and  on  agar  the  green  colour 
diffuses  into  the  substance  of  the  medium.  Acid  and 
gas  are  produced  in  the  majority  of  the  carbohydrate 
media. 

B.  pyocyaneus  most  frequently  occurs  as  a  secondary 
infection,  often  in  association  with  lesions  connected  with  the 
intestinal  tract,  as  in  ischio-rectal  abscess  or  in  general  perito- 
nitis secondary  to  a  gangrenous  appendix.  The  bacillus  may  also 
be  associated  with  cerebral  abscess,  a  spreading  cellulitis,  or  a 
local  abscess.  The  pus  in  these  infections  is  alleged  to  be  of  a 
sky-blue  colour,  a  tint  which  is  far  from  obvious.  The  lesions 
associated  with  B.  pyocyaneus  are  often  somewhat  intract- 
able, and  a  general  peritonitis  accompanied  by  this  bacillus  is 
almost  invariably  fatal.  After  inoculation  into  the  peritoneal 
cavity  of  a  guinea-jiig  a  virulent  and  rapidly  fatal  peritonitis 
follows. 

The  bacillus  of  malignant  (Edema. — This  bacillus  is 
commonly  somewhat  larger  than  the  other  members  of  this 
group,  is  sluggishly  motile,  and  forms  centrally  situated 
spores.  The  organism  grows  readily,  but  only  under  anaerobic 
conditions.  It  produces  abundant  gas  in  the  carbohydrate 
media.  The  bacillus  is  widely  distributed  in  nature,  and 
may  be  found  in  many  samples  of  garden  earth.  It  rarely 
gains  entrance  into  the  human  body,  but  may  obtain  a 
footing  in  a  septic  wound  and  set  up  a  virulent  spreading 
gangrene,  associated  with  the  formation  of  blebs  and  the 
production  of  gas. 

The  Spieilla. 

The  spirilla  form  an  important  group  of  organisms  which, 
owing  to  the  inclusion  of  the  cholera  vibrio,  differ  widely  from 
each  other.  The  cholera  vibrio  is  an  organism  which  cul- 
turally resembles  the  members  of  the  coli -typhoid  group,  and, 
being  merely  a  curved  rod  and  not  a  true  spiral,  might  reason- 
ably be  classed  among  the  bacilli.  The  properly  spiral 
organisms,  which  include  the  spirochete  of  syphilis,  have 
numerous  points  of  resemblance,  and  are  evidently  possessed 
of  a  higher  organisation  than  the  bacilli.  They  are  included 
by  many  authorities  among  the  protozoa. 

The  cholera  vibrio  (Comma  bacillus)  (Plate  IX.). — The 
cholera  spirilla  are  small,  actively  motile,  flagellated  curved 


GEAM-NEGATIVE   BACILLI— SPIRILLA,  ETC.     141 

rods,  having  a  tendency  to  lie  with  their  long  axes  in  the 
same  direction.  The  spirilla  grow  readily  on  all  the  ordinary 
media.  They  liquefy  gelatin,  and  the  young  colonies  on 
gelatin  plate  cultures  are  irregularly  granular  and  look  like 
"fragments  of  broken  glass."  In  broth  a  pellicle  is  produced 
on  the  surface  of  the  medium,  and  both  indole  and  a  nitrite  are 
formed  from  the  peptone,  so  that  a  pink  colour  (the  cholera- 
red  reaction)  appears  on  the  addition  of  a  few  drops  of  sulphuric 
acid  only.  Litmus  milk  is  unchanged.  The  identification  of 
the  organism  from  similar  vibrios  should  be  completed  by 
testing  the  lytic  action  of  immune  serum  upon  it  on  the  lines 
indicated  in  Chapter  VIII. ,  page  110. 

In  the  human  body  the  organisms  are  confined  to  the 
intestine,  and  in  the  acute  cases  the  watery  stools  appear  to 
form  an  almost  pure  culture  of  the  organism.  Numerous 
other  varieties  of  spirilla  have  been  described,  and  are  of 
particular  interest  in  that  they  may  be  mistaken  for  the  cholera 
vibrio.  Metchnikoff  has  recorded  a  similar  vibrio  found  in 
the  intestines  of  fowls  dead  of  gastro-enteritis.  Einkler 
and  Prior  described  another  present  in  infantile  diarrhoea. 
Vibrios  distinct  from  the  cholera  spirillum  have  been  found 
also  in  the  mouth,  and  in  the  water  supply  of  numerous 
towns.  It  is  evident,  therefore,  that  considerable  caution 
should  be  exercised  in  the  identification  of  the  genuine  vibrio, 
particularly  in  the  absence  of  an  epidemic. 

The  Spirochaeta  Pallida  (Treponema  Pallidum)  (Plate  IX.). 
— In  the  fresh  exudate  examined  under  the  dark  ground 
illumination  (page  161)  the  spirochete  of  syphilis  appears  as 
a  slowly-moving,  extremely  delicate  organism  with  numerous 
regular  spirals,  which  do  not  straighten  out  during  rest.  On 
occasion  small  refractile  granules  (the  granular  bodies  of 
Leishman)  may  be  seen  to  be  shot  out  from  the  spirochete 
into  the  surrounding  medium.  In  Indian  ink  preparations 
(page  160)  the  spirochete  has  a  very  similar  appearance,  but 
has  of  course  no  movement.  The  spirochsete  is  left  unstained 
by  the  ordinary  methods,  and  is  best  stained  by  Giemsa's  dye 
after  preliminary  fixation  with  absolute  alcohol  (page  160). 
The  appearance  in  stained  preparations  is  very  characteristic. 
The  Spiroclueta  pallida  stains  pink,  in  contradistinction  to  the 
majority  of  other  spirochetes,  which  stain  a  bluish  red.  The 
minute  delicate  spirals  are  numerous — from  10  to  20 — and  very 


142  CLINICAL   PATHOLOGY. 

regular.  The  spirochete  is  usually  present  in  considerable 
numbers  in  both  primary  and  secondary  lesions.  It  has  been 
very  rarely  demonstrated  in  tertiary  syphilis  and  never  in 
parasyphilis.  The  Spirochceta  pallida  cannot  be  cultivated  on 
the  usual  media.  Inoculation  of  fluid  containing  the  organisms 
into  one  of  the  higher  apes  is  followed  by  a  local  chancre 
and  later  by  secondary  manifestations.  Inoculation  into  the 
lower  monkeys  or  into  the  testicle  of  a  rabbit  is  followed  by  a 
local  sore  only,  which  tends  to  heal  spontaneously. 

The  early  diagnosis  of  syphilis  by  the  demonstration  of  the 
spirochete  in  the  primary  chancre  before  the  serum  reaction 
has  become  positive  is  of  the  utmost  importance,  since  the 
cure  of  the  disease  depends  so  much  upon  the  promptitude 
with  which  treatment  is  commenced.  The  success  of  the 
demonstration  rests  largely  upon  the  care  with  which  the 
material  to  be  examined  is  obtained.  The  surface  of  the 
chancre  should  first  be  washed  over  thoroughly  with  sterile 
salt  solution  and  an  attempt  made  by  vigorous  squeezing 
of  the  edges  of  the  ulcer  to  cause  a  clear  or  blood-tinged 
serum  to  exude  from  the  depths  of  the  base.  If  the  chancre 
is  very  painful  or  firmly  encrusted  over,  the  surface  should  be 
well  swabbed  with  4  per  cent,  eucaine  and  a  further  attempt 
made  to  obtain  the  proper  fluid,  aided,  if  necessary,  by  a 
scraping  of  the  chancre  base  with  the  edge  of  a  glass  micro- 
scope slide.  The  fluid  is  transferred  in  a  platinum  loop  to  a 
slide,  and  both  stained  and  fresh  specimens  should  be  pre- 
pared (page  160).  The  greatest  care  should  be  taken  to  avoid 
infecting  one's  own  fingers  in  performing  this  examination 
and  to  wash  immediately  and  thoroughly  if  the  fingers  touch 
the  chancre. 

A  negative  examination  is  not  sufficient  contra-indication  of 
syphilis,  but  in  skilled  hands  negative  results  are  unusual.  In 
cases  of  doubt  a  second  examination  should  be  made,  and  if 
enlarged  glands  are  present  in  the  groin  the  most  prominent 
should  be  punctured  with  a  hypodermic  needle,  and  the 
minute  quantity  thus  obtained  often  yields  a  positive  result. 
The  recognition  of  the  Spirochceta  pallida  is  positive  evidence 
of  syphilis,  provided  the  identification  of  the  organism  is 
correct.  Numerous  other  varieties  of  spiral  organisms  are  to 
be  found  in  the  body,  particularly  in  the  region  of  the  mouth,  the 
anus,  and  much  less  frequently  the  male  urethra  and  the  vulva. 


GRAM-NEGATIVE    BACILLI— SPIRILLA,  ETC.     143 

Particular  care,  therefore,  should  be  exercised  in  the  examina- 
tion of  the  majority  of  extragenital  chancres.  The  main 
points  in  the  identity  of  the  syphilitic  organism  are :  the  com- 
parative lack  of  motility  on  the  dark  ground  illumination,  the 
failure  to  stain  by  the  ordinary  dyes  and  the  rose-pink  colour 
when  stained  by  Giemsa's  dye,  the  delicacy  of  the  spirochete, 
the  large  number  of  its  spirals  (from  12  upwards  should  be 
counted)  and  their  regularity.  The  more  important  of  the 
spirochetes  which  can  be  confused  with  S.  pallida  are  men- 
tioned below. 

The  Spirochete  of  Yaws  (S.  pertenuis)  is  morpho- 
logically identical  with  S.  pallida. 

Spirochseta  Refringens,  etc.— 8.  refringens  is  a  sapro- 
phytic organism,  and  is  frequently  associated  with  S.  pallida. 
It  is  long,  coarse,  and  has  about  six  irregular  spirals.  It 
stains,  though  somewhat  faintly,  with  the  ordinary  dyes. 

S.  Gracilis  occupies  an  intermediate  position,  so  far  as 
appearance  goes,  between  S.  pallida  and  S.  refringens. 

S.  Dentium  is  a  minute  spirochete,  of  about  half  the  length 
of  S.  pallida,  and  is  commonly  found  in  the  mouth.  It 
stains  with  the  ordinary  dyes. 

Many  other  names  have  been  given  to  these  saprophytic 
spirilla,  some  of  which  have  been  cultivated,  and  a  useful 
study  of  some  of  them  can  easily  be  made  by  examining  films 
made  from  any  case  of  ulcerative  stomatitis,  or  even  from  a 
comparatively  normal  mouth,  by  the  methods  used  in  the 
diagnosis  of  the  S.  pallida. 

The  spirillum  and  fusiform  bacillus  of  Vincent 
(Plate  IX.). — Among  the  spirochetes  which  have  achieved 
notoriety  either  as  producing  disease  or  as  saprophytes  the 
organisms  of  Vincent  must  be  mentioned.  These  organisms 
consist  of  spirilla  which  are  usually  large,  irregular,  and  with 
few  spirals,  resembling  S.  refringens,  and  less  commonly 
minute  and  delicate  like  S.  dentium.  The  spirilla  are  asso- 
ciated in  film  preparations  with  large,  coarse,  fusiform  bacilli. 
Both  organisms  stain  with  the  ordinary  dyes  and  do  not 
grow  on  the  ordinary  media.  The  spirilla  and  bacilli  are 
fairly  constantly  present  in  septic  mouths.  They  are 
extremely  numerous  in  certain  forms  of  ulcerative  stomatitis 
and  tonsilitis,  often  associated  with  sloughing  and  membrane 
formation,  particularly  in  children.      The  diphtheria  bacillus 


144  CLINICAL  PATHOLOGY. 

is  absent  in  such  cases,  though  other  organisms,  such  as 
streptococci,  are  commonly  present.  The  condition  is  known 
as  Vincent's  angina. 

Spirochaeta  Obermeieri  (S.  recurrentis). — This  organism 
has  been  considered  in  the  chapter  on  the  parasitology  of  the 
blood. 

StreptotrichejE. 

Actinomyces  (Ray  fungus)  (Plate  IX.).— This  class  of 
organism,  which  doubtless  embraces  a  number  of  closely- 
allied  species,  rarely  attacks  man,  and  is  found  more  commonly 
among  the  domestic  animals. 

Under  the  microscope  the  parasite  is  found  to  consist  of 
three  parts: — (1)  Long,  thin  filaments  enclosed  in  a  sheath 
and  often  beaded  :  the  filaments  are  interlaced  and  may  show 
branching;  they  are  Gram-positive.  (2)  Coccus-like  bodies, 
formed  from  the  filaments,  and  which  have  been  considered 
to  represent  gonidia ;  they  also  are  Gram-positive.  (3)  Clubs 
or  elongated  pear-shaped  bodies  radiating  from  the  centre  of 
an  actinomycotic  colony ;  they  are  usually  Gram-negative. 

Actinomyces  grow  on  all  the  ordinary  media,  and  on 
glycerin-agar  growth  becomes  visible  about  the  fourth  day, 
and  later  forms  tough,  nodular,  granulated  colonies  of  a 
brownish  colour.  Gelatin  is  liquefied.  Clubs  are  not  present 
in  cultures. 

In  the  human  subject  actinomyces  may  attack  the  mouth  or 
tongue,  spreading  from  thence  to  the  glands  of  the  neck,  or 
may  effect  a  lodgment  in  the  intestine,  and  not  infrequently 
in  the  appendix,  and  spread  from  thence  to  the  liver  and 
other  parts  of  the  body. 

In  the  examination  of  pus  from  suspected  actinomycotic 
lesions  the  little  colonies  which  are  visible  to  the  naked  eye 
should  be  sought  for.  They  are  readily  recognised  as  round 
or  oval  greenish-yellow  granules  of  about  the  size  of  a  pin's 
head,  more  or  less  translucent  and  definitely  resistant  to 
the  touch.  If  a  granule  is  squashed  on  a  slide  and  stained  by 
carbol  thionin  or  by  Gram's  method  the  beaded  filaments  can  be 
certainly  recognised.  The  clubs,  which  are  common  in  lesions 
of  the  ox,  are  very  rarely  seen  in  man.  Cultures  are  not 
strictly  necessary  for  diagnosis,  and  are  often  difficult  to 
obtain   owing    to   the   presence   of    other   organisms.      The 


GEAM-NEGATIVE   BACILLI— SPIRILLA,   ETC.     145 

granules  should  be  picked  out  with  a  sterile  needle  and 
repeatedly  washed  in  sterile  salt  solution  before  planting  on 
glycerin-agar. 

Other  varieties  of  streptothrices  have  been  described  and 
isolated  in  lesions  from  human  beings  which  differ  in  their 
cultural  characters,  and  particularly  in  the  colour  of  their 
colonies,  on  solid  media.  Some  are  acid-fast,  and  the  majority 
of  them  produce  caseous  nodules  when  inoculated  into 
guinea-pigs.  These  streptothrices  are  rarely  met  with,  but 
may  produce  pulmonary  lesions  such  as  can  be  mistaken  on 
clinical  grounds  for  tuberculosis,  and  on  pathological  grounds 
may  escape  identification  if  the  beaded  threads  are  acid-fast. 
Suspected  cases  should  be  carefully  examined  both  micro- 
scopically and  culturally,  and  the  sputum  should  be  inoculated 
into  guinea-pigs  with  a  view  to  recovering  the  organism  in 
pure  culture. 

Madura  disease  is  comparatively  common  in  India  and 
in  other  tropical  countries.  It  is  a  granulomatous  con- 
dition of  the  foot  set  up  by  a  streptothrix  culturally 
distinct  from  actinomyces.  The  growth  is  pigmented,  and 
the  colour  may  be  either  pale  pink  or  black.  The 
mycelial  filaments  and  the  granules  may  readily  be  detected 
in  film  preparations  made  from  colonies  scraped  out  of 
the  skin. 

Hyphomycetes. 

The  more  important  of  the  hyphomycetes  are  in  human 
pathology  met  with  in  diseases  of  the  hair  and  skin.  A  brief 
description  only  can  be  given  here.  The  organisms  are  again 
referred  to  in  the  section  on  the  skin. 

Ringworm. — Two  species  of  parasite  are  met  with.  The 
microsporon  Audouini  is  the  common  cause  of  ringworm 
among  children,  and  appears  in  the  form  of  numerous  small 
spores  and  scanty  mycelial  threads  situated  outside  the  affected 
hairs. 

Trichophyton  megalosporon  endothrix  is  less  fre- 
quently met  with;  the  spores  are  large  and  the  parasite  is 
situated  within  the  hair  shaft. 

The  majority  of  such  moulds  can  be  grown  on  agar  media 
and  preferably  on  Sabouraud's  maltose  agar  medium. 

p.  10 


146  CLINICAL   PATHOLOGY. 

The  demonstration  of  the  parasites  in  hair  can  be  performed 
as  follows  : — 

Eemove  with  a  pair  of  fine  forceps  a  few  of  the  bent  and 
stunted  hairs. 

Place  in  a  watch-glass  and  soak  in  ether  for  a  few  minutes. 

Mount  on  a  slide  in  10  per  cent,  caustic  potash  beneath  a 
cover-glass  and  examine  with  a  J-inch  objective  for  the  oval 
refractile  spores. 

To  make  a  permanent  specimen  : — 

After  cleansing  in  ether  fix  on  a  slide  with  a  little  melted 
paraffin. 

Stain  by  Gram's  method  up  to  the  stage  of  decolorising  in 
methylated  spirit,  which  should  be  completed  by  immersion  in 
absolute  alcohol.  Then  wash  in  xylol,  and  scrape  off  the 
remainder  of  the  paraffin.  Drain  off  the  xylol  and  mount 
in  Canada  balsam. 

Favus  is  due  to  a  parasite,  the  Achorion  schoenleinii,  which 
consists  of  a  tubular  branching  mycelium  with  a  few  oval 
spores. 

Pityriasis  versicolor  is  caused  by  the  Microsporon  furfur, 
which  consists  of  abundant  mycelium,  spores  and  hyphse  and 
is  readily  cultivated. 

To  examine  for  these  parasites  scrapings  can  be  taken  of  the 
affected  skin  scales  and  mounted  in  potash  or  stained  with 
gentian  violet  in  a  similar  manner  to  the  examination  of  hairs 
for  ringworm. 

Aspergillus  niger  is  an  instance  of  a  non-pathogenic 
mould  which  may  on  rare  occasions  infect  the  tissues,  and  has 
been  found  widely  distributed  throughout  the  lungs.  The 
organism  produces  a  sooty  growth  on  potato. 

Thrush  (Plate  IX.)  is  caused  by  the  Oidium  albican?,  an 
organism  classed  by  some  among  the  moulds,  by  others  among 
the  blastomycetes  or  yeasts.  It  grows  like  a  yeast  in  culture, 
showing  large  oval  budding  cells,  and  in  scrapings  from  the 
throat  coarse  mycelial  threads  are  seen,  together  with  as  a  rule 
the  yeast-like  cells. 

The  Protozoa. — The  majority  of  these  have  been  described 
in  the  chapter  on  the  parasitology  of  the  blood.  Others,  such 
as  the  Entamoeba  histolytica  of  tropical  dysentery,  are  con- 
sidered under  the  examination  of  tbe  fceces. 


CHAPTER   XL 

bacteriological  methods — general  and  special. 
General  Methods. 

The   general  apparatus   required   for   the  majority  of 
bacteriological  examinations  consists  of — 
Slides  and  cover-glasses. 
Platinum  wire. 
Forceps. 

Culture  tubes  and  crates. 
Incubator. 
Staining  reagents. 

The  slides  and  cover-glasses  used  require  no  special  pre- 
paration, but  are  conveniently  kept  in  methylated  spirit  in 
wide-mouthed  glass  jars  until  required  for  use,  when  they  are 
wiped  dry  with  a  clean  cloth.  After  use  the  slides  can  be 
placed  in  lysol  for  an  indefinite  time  and  subsequently  cleaned 
and  used  again. 

The  platinum  wire  should  be  a  moderately  stout  piece  about 
4  inches  long.  The  wire  should  be  fixed  into  one  end  of  a 
glass  rod  about  9  inches  in  length  by  the  simple  procedure  of 
heating  the  end  of  the  rod  to  red  heat  in  the  blow-pipe  flame 
and  plunging  the  platinum  wire  into  it  while  hot.  The  free  end 
of  the  platinum  wire  should  then  be  bent  round  upon  itself  to 
form  a  small  oval  loop.  The  returning  end  of  the  wire  should 
exactly  meet  the  straight  wire  and  should  not  overlap  it.  An 
untidy  platinum  wire  is  often  responsible  for  untidy  and  con- 
sequently both  inaccurate  and  dangerous  bacteriological  work. 

The  forceps  should  be  of  the  ordinary  straight  variety  used 
in  dissection  or  operation  work.  It  is  convenient  also  to 
have  a  pair  of  catch  forceps  of  the  pattern  known  as  Cornet's. 
The  culture  tubes  should  be  of  the  kind  described  in  a  former 
chapter,  and  can  be  bought  ready  for  use.  If  the  amount 
of  bacteriological  work  required  is  considerable,  it  is  very 
much  more  satisfactory  and  economical  to  prepare  the  media 
in  the  laboratory.     The  methods  of  making  the  media  will  be 

10—2 


148 


CLINICAL  PATHOLOGY. 


described  subsequently.  The  essential  media  consist  of  broth 
and  agar  slopes,  and  these  are  sufficient  for  taking  the  primary 
cultures  in  most  cases.  For  the  complete  investigation  of 
organisms  other  media  are  necessary.  After  inoculation  the 
tubes  are  preferably  placed  in  basket  wire  crates  lined  at  the 
bottom  with  cotton  wool.  The  crates  can  be  dispensed  with 
and  ordinary  glass  beakers  or  even  old  coffee  tins  may  be 
made  use  of  instead. 

The  incubator  should  be  one  capable  of  maintaining  a 
constant  temperature  of  37°  C.  Reliable  incubators  pro- 
vided with  self-regulating   capsules   are  supplied   by  several 

firms.  A  less  expensive  ap- 
paratus consists  of  a  copper 
chamber  fitted  with  a  water- 
jacket  and  warmed  by  a  gas-jet, 
the  heat  within  the  chamber  being 
controlled  by  the  size  of  the  jet. 
Such  an  apparatus  is  liable  to  vary 
with  any  considerable  fluctua- 
tions in  the  room  temperature. 

"When  occasional  bacteriological 
investigations  only  are  required, 
a  wide-mouthed  "Thermos"  flask 
can  be  readily  adapted.  The 
variety  of  flask  made  to  hold 
soups  or  stews,  and  costing  30s., 
is  the  most  suitable,  and  can  be 
fitted  with  an  amateur  copper 
wire  framework  capable  of  hold- 
ing 4  culture  tubes.  The  flask  is  filled  about  two-thirds  fall  with 
water  at  40°  C,  a  temperature  which  in  18  hours  will  have 
dropped  to  35°  C.  Such  an  incubator  is  sufficiently  reliable 
for  the  cultivation  of  diphtheria  bacilli,  or  indeed  the  majority 
of  readily-growing  pathogenic  organisms. 

The  staining  reagents  requisite  for  all  ordinary  bacterio- 
logical work  are  few  in  number.  The  majority  of  them  can 
be  bought  ready  made  up  in  solution,  and  the  mode  of  pre- 
paration of  each  is  given  subsequently. 

The  general  procedure  to  be  followed  when  investigating 
the  bacterial  content  of  pus  or  of  any  body  fluid  is  as  follows  ; — 
(1)  Make  films  of  the  pus. 


Fig.  11. — Bacteriological 
Incubator. 


BACTERIOLOGICAL   METHODS.  149 

((a)  with  some  simple  dye    such  as  carbol- 

(2)  Stain  themj  thionin. 

( (b)  by  Gram's  method. 

(3)  Examine  the  films. 

(4)  Put  up  cultures  in  both  liquid  and  solid  media. 

(5)  Incubate  at  37°  C.  for  from  12  to  24  hours. 

(0)  Examine  the  culture  tubes  with  the  naked  eye  and  with 

a  hand-glass. 

(7)  Make  films  from  the  cultures.    Stain  and  examine  them. 

(8)  If  the  organism  is  in  pure  culture,  subculture  it  from 

the  solid  medium  into  the  appropriate  media.     If  a 

variety  of  organisms  are  present  plate  out  from  the 
culture  into  Petri  dishes. 

(9)  After   incubation  of   the  sub-cultures  for  24  hours  (or 

longer  if  necessary)  examine  them,  note  the  changes 
which  have  occurred  in  them,  and  make  film  pre- 
parations from  them. 

(1)  To  make  films  from  pus. — Clean  a  slide.  Sterilise 
the  platinum  wire  by  heating  it  to  a  red  heat  in  a  Bunsen 
flame.  Let  the  wire  cool  in  the  air.  Take  up  a  loop  of  the 
pus  and  spread  it  evenly  by  a  circular  motion  so  as  to  make  a 
thin  round  film  about  the  size  of  a  shilling  in  the  centre  of 
the  slide.  Sterilise  the  platinum  wire.  Dry  the  film  by 
waving  it  in  the  air  above  the  flame.  Fix  by  passing  it  three 
times  rapidly  through  the  flame. 

(2)  To  stain  the  films.—  (a)  With  a  simple  stain.  This 
should  always  be  done  as  a  routine.  The  examination  of  films 
made  from  the  original  fluid  yields  information  which  may  be 
altogether  missed  in  the  investigation  of  the  culture  tubes. 
Also  an  indication  is  obtained  of  the  nature  of  the  organism 
and  consequently  of  any  special  stain  that  may  be  necessary, 
as  well  as  of  the  appropriate  media  upon  which  to  make  the 
cultures. 

The  best  general  stain  is  carbol-thionin.  The  advantages 
of  this  stain  are  that  it  brings  out  the  majority  of  organisms 
and  the  cells  present,  and  that  it  is  almost  impossible  to  either 
under-  or  over-  stain  the  films. 

To  stain  with  carbol-thionin.— Cover  the  entire  slide 
with  the  stain  ;  do  not  merely  place  a  few  drops  of  the  stain 
on  the  film  itself. 

Leave  for  approximately  3  minutes. 


150  CLINICAL   PATHOLOGY. 

Wash  in  tap  water.     Blot  dry.     Mount  in  Canada  balsam. 

(The  mounting  of  any  film  which  is  to  be  examined  with  a 
Y^-inch  objective  is  unnecessary  unless  a  permanent  specimen 
is  required,  since  the  cedar  oil  placed  directly  on  the  film 
clears  it.) 

In  place  of  carbol-thionin,  dilute  methylene  blue  may  be 
used ;  the  staining  process  is  identical. 

(b)  By  Gram's  method. — It  is  not  necessary  to  make 
use  of  this  method  in  all  cases.  The  less  experience  the 
student  has  in  the  examination  of  bacteriological  films  the 
more  frequently  he  should  take  advantage  of  the  information 
given  by  Gram's  stain. 

To  stain  by  Gram's  method. — Take  3  c.c.  of  aniline  oil 
and  17  c.c.  of  distilled  water  in  a  measure  glass.  Shake 
vigorously  for  5  minutes.  Filter.  The  filtrate  must  be  clear. 
Take  3  c.c.  of  a  saturated  solution  of  gentian  violet  in  absolute 
alcohol  and  7  c.c.  of  the  aniline  water.     Mix. 

Filter  the  aniline  gentian  violet  on  to  the  slide. 

Stain  for  5  minutes. 

Pour  off  the  stain  and  rapidly  dip  the  slide  in  tap  water. 
Drain  off  the  water. 

Cover  the  slide  with  Gram's  iodine. 

Leave  for  30  seconds. 

Wash  in  methylated  spirit  and  continue  to  wash  until  the 
blue  colour  ceases  to  run  from  the  film. 

Blot  dry. 

Countersfcain  with  carbol-fuchsin  diluted  with  tap  water  to 
about  the  colour  of  red  ink. 

Stain  for  2  minutes. 

Wash  in  tap  water.     Blot  dry.     Mount. 

The  principle  of  the  stain  is  as  follows.  Aniline  gentian 
violet  stains  the  great  majority  of  all  organisms.  If  the  film 
is  then  washed  in  spirit  the  stain  comes  out  again.  But  if 
the  film,  after  staining,  is  treated  with  Gram's  iodine  the 
gentian  violet  is  fixed  in  some  organisms  so  as  to  resist 
subsequent  decoloration  by  spirit,  but  not  in  other  organisms. 
The  counter-stain  with  carbol-fuchsin  is  not  part  of  Gram's 
method,  but  is  used  to  display  those  organisms  which  have 
lost  their  colour  in  the  spirit.  "  Gram-positive  "  organisms 
are  consequently  coloured  violet  and  "  Gram-negative  " 
organisms   red.     The   distinction    between   these   colours   is 


BACTERIOLOGICAL  METHODS.  151 

more  obvious  by  daylight  than  by  artificial  light.  No  fixed  time 
is  given  for  the  decoloration  stage  in  spirit,  because  the  time 
occupied  depends  largely  upon  the  thickness  of  the  film.  It 
must  be  recognised  that  it  is  possible  to  wash  the  colour  out 
of  a  "Gram-positive"  organism  or 'to  leave  the  colour  in  a 
"  Gram-negative  "  one  if  the  washing  is  too  long  or  too  short. 
The  alcohol  process  is  finished  directly  the  blue  colour  ceases 
to  run  from  the  film,  and  this  point  is  readily  determined  if 
a  small  quantity  of  clean  methylated  spirit  is  reserved  for  the 
final  dipping  of  the  slide. 

(3)  To  examine  the  films.— Use  a  No.  2  eye-piece,  a 
Y^-inch  objective  and,  when  available,  daylight. 

Look  for  the  kind  of  cell  present.  In  the  great  majority  of 
such  films  these  will  be  polynuclears,  with  an  occasional  large 
hyaline  cell  and  possibly  a  few  epithelial  cells. 

Examine  for  the  presence  of  bacteria,  and  note  whether 
they  are  cocci  or  bacilli,  whether  they  are  arranged  in  pairs, 
clumps  or  chains,  and  whether  they  are  mainly  within  the 
cells  or  outside  them.  If  more  than  one  variety  of  organism 
is  evidently  present  observe  which  variety  appears  to  pre- 
dominate. If  no  bacteria  are  seen  in  the  films  the  causative 
organism  may  still  be  recovered  in  the  cultures. 

(4)  To  put  up  cultures. — If  the  pus  is  taken  directly 
from  the  body  it  is  commonly  necessary  to  make  both  films  and 
cultures  at  the  same  time.  In  such  cases  the  culture  media 
suitable  to  the  suspected  organism  are  chosen.  If  the  pus  is 
received  in  a  sterile  receptacle  in  the  laboratory  it  is  preferable 
to  examine  the  films  before  putting  up  the  cultures.  In  the 
majority  of  cases  two  tubes  should  be  inoculated,  and  these 
should  be  a  broth  tube  and  an  agar  slope. 

It  is  often  convenient  to  add  a  litmus  milk  tube  or  to 
substitute  it  for  the  broth  tube,  since  some  organisms,  and  in 
particularly  the  pneumococcus,  grow  more  readily  in  milk 
than  in  broth  or  even  than  on  agar.  When  organisms  such 
as  the  gonococcus  or  meningococcus,  which  do  not  grow  at  all, 
or  grow  poorly,  in  the  primary  cultures  on  agar  and  broth,  are 
suspected  the  appropriate  blood-containing  media  must  be 
used. 

To  inoculate  the  tubes. — Hold  the  test  tube  containing 
the  pus  and  the  culture  tubes  between  the  thumb  and  first 
finger  of  the  left  hand.     The  tubes  should  not  be  held  upright, 


152  CLINICAL   PATHOLOGY. 

but  in  a  slanting  direction,  since  dust  and  organisms  may 
fall  into  them. 

Sterilise  the  platinum  loop  in  the  flame.  Allow  it  to  cool. 
Place  the  glass  handle  between  the  first  and  second  fingers 
of  the  left  hand. 

Take  a  pair  of  forceps  in  the  right  hand  and  with  a 
screwing  motion  twist  out  the  cotton-wool  plugs  from  the 
tubes.  Place  each  plug  between  the  third  and  little  fingers 
of  the  left  hand. 

Put  down  the  forceps  on  the  bench. 

Take  the  platinum  wire  in  the  right  hand  and  pass  it  into  the 
tube  of  pus.  Take  out  the  wire,  pass  it  into  the  agar  slope 
tube  and  spread  it  gently  over  the  surface  of  the  medium. 
Dip  the  wire  again  into  the  pus,  then  into  the  broth  tube,  and 
shake  off  the  pus  into  the  broth.  Be  careful  not  to  touch  the 
sides  of  any  of  the  tubes  with  the  wire.  Sterilise  the  wire 
and  lay  it  down. 

Pick  up  the  forceps.  Take  each  wool  plug  separately  and 
light  it  in  the  flame.  Put  the  plug,  still  alight,  into  the  tube. 
Do  not  blow  the  plug  out ;  it  will  cease  to  burn  as  soon  as  it  is 
pressed  well  home  in  the  tube.  With  a  blue  glass-pencil 
mark  each  tube  with  the  date  and  a  distinguishing  number. 

(5)  To  incubate  the  tubes. — Place  the  tubes  in  a  wire 
crate,  taking  care  not  to  knock  them  on  the  edge  of  the  crate, 
and  place  in  the  incubator  at  37°  C.  till  the  following  day. 
From  12  to  24  hours  is  sufficient  time  in  which  to  obtain 
a  naked-eye  growth  of  the  majority  of  organisms,  but  the 
cultures  should  not  be  considered  sterile  before  the  lapse  of 
at  least  4  to  5  days.  Exceptional  organisms,  such  as  the 
tubercle  bacilli,  show  little  or  no  growth  before  the  tenth 
day. 

(6)  To  examine  the  culture  tubes. — Remove  the  tubes 
from  the  incubator  and  look  at  them  to  see  if  any  growth  has 
taken  place.  Note  the  character  of  the  growth  in  broth  and 
the  size,  shape,  colour,  and  density  of  the  colonies  on  the 
agar  slope.  Examine  the  agar  slope  further  with  a  hand 
lens. 

(7)  To  make  films  from  the  cultures. — In  the  case  of 
a  liquid  medium  hold  the  tube  in  the  left  hand,  sterilise  the 
platinum  wire,  pass  it  into  the  left  hand,  and  allow  it  to  cool. 
Remove  the  wool  plug  with  forceps  and  with  the  wire  take 


BACTERIOLOGICAL   METHODS.  153 

up  a  considerable  loop  of  the  broth  (or  milk)  culture.  Pass 
the  wire  back  into  the  left  hand.  Ignite  the  plug  and  replace 
it  in  the  tube.  Make  as  thick  a  film  as  possible  exactly  in 
the  centre  of  a  clean  slide.     Sterilise  the  wire. 

In  the  case  of  a  solid  medium  first  place  a  small  drop  of 
tap  water  on  the  centre  of  the  slide,  then  proceeding  as 
above,  take  up  in  the  wire  a  small  particle  of  the  growth  and 
rub  it  in  the  water,  making  as  thin  a  film  as  possible.  Allow 
the  films  to  dry.  Fix  them  in  the  flame.  Stain  with  carbol- 
thionin. 

If  necessary  make  additional  films  and  stain  by  Gram's 
method.     Examine  the  films  under  the  microscope. 

(8)  To  make  sub-cultures. — It  is  advisable  in  the 
majority  of  cases  to  "  plate  out  "  from  the  broth  culture,  but 
this  may  be  dispensed  with  if  an  examination  of  the  original 
films  and  of  the  cultures  reveals  only  one  variety  of  organism. 
Particular  attention  is  to  be  paid  to  the  nature  of  the  colonies 
on  the  agar  slope,  and  if  these  appear  identical  with  each 
other  the  culture  may  be  presumed  "pure." 

When  the  original  cultures  are  pure,  subculture  from  the 
agar  slope  into  those  media  which  give  characteristic  reactions 
with  the  suspected  organism.  In  most  cases  it  is  advisable 
to  subculture  into  litmus  milk,  neutral  red  broth,  a  selection 
of  the  litmus  carbohydrate  broths,  and  on  to  gelatin  slopes. 

To  make  the  sub-culture  hold  the  agar  tube  and  two  or 
three  of  the  tubes  to  be  inoculated  in  the  left  hand.  Sterilise 
the  wire.  Allow  it  to  cool.  Scrape  off  a  colony,  or  portion  of 
a  colony,  from  the  agar  slope.  Shake  off  the  growth  in  a 
liquid  medium  or  rub  it  over  the  surface  of  a  solid  one. 
Sterilise  the  wire.  Replace  the  plugs.  Label  each  sub-culture 
with  the  name  of  the  medium  (particularly  in  the  case  of  the 
carbohydrate  media),  the  date,  and  the  name  or  number  of 
the  case. 

A  variant  of  the  above  procedure  consists  in  taking  a  single 
colony  from  the  agar  slope  and  inoculating  it  upon  a  second 
agar  slope,  incubating  till  the  next  day,  and  then  subculturing 
from  the  second  slope  into  the  media.  By  this  method  a  pure 
culture  is  practically  assured,  but  an  extra  day  is  required. 

When  the  original  cultures  are  "  mixed  "  it  is  necessary  to 
separate  the  different  organisms,  and  this  is  done  by  means  of 
plate   cultures   in  Petri   dishes.     A   minimum  of   two  plates 


154  CLINICAL   PATHOLOGY. 

should  be  used,  and  if  the  organisms  are  varieties  of  cocci  both 
plates  should  contain  agar.  If  organisms  of  the  colon  group 
are  suspected  prepare  one  agar  plate  and  one  plate  containing 
MacConkey's  neutral  red  medium.  The  plate  cultures  are  pre- 
pared as  follows  : — Place  "  stab  "  culture  tubes  in  a  tall  beaker 
filled  with  hot  water  to  above  the  level  of  the  medium  in  the 
tubes.  Bring  the  water  to  the  boiling  point  over  a  Bunsen 
burner.  When  the  media  are  completely  liquid  turn  out  the 
gas  and  leave  for  5  or  10  minutes.  Have  ready  sterile  Petri 
dishes  with  well-fitting  lids.  Take  a  melted  stab  culture  tube 
from  the  beaker.  Twist  out  the  wool  plug  with  forceps.  Pass 
the  mouth  of  the  test  tube  through  the  flame  (to  sterilise  the 
outside  of  the  glass).  Lift  ivp  the  lid  of  the  Petri  dish  just 
enough  to  insert  the  mouth  of  the  test  tube.  Pour  the  con- 
tents of  the  test  tube  into  the  dish.  Replace  the  lid  and  rotate 
the  dish  so  as  to  spread  the  medium  evenly  over  its  bottom. 
As  soon  as  the  medium  is  set,  place  the  dish  upside  down  in 
the  ice-chest  (or  failing  that  at  room  temperature)  until  the 
dish  feels  quite  cold.  To  use  the  plates  : — Take  the  incubated 
broth  culture  tube  in  the  left  hand.  Sterilise  the  platinum 
wire.  Take  one  loop  of  the  broth  culture.  Put  back  the  broth 
tube  in  the  crate  after  replacing  the  plug.  Lift  up  the  lid  of 
the  agar  plate  the  minimum  distance.  Rub  the  platinum  wire 
repeatedly  across  the  agar  medium  in  a  succession  of  parallel 
streaks.  Replace  the  lid  of  the  dish.  Lift  the  lid  of  the 
second  agar  or  the  MacConkey  plate  and  repeat  the  process 
without  recharging  the  platinum  loop. 

Place  the  plates  in  the  incubator  upside  down  in  order  to 
prevent  the  water  of  condensation  wThich  collects  in  the  lid  of 
the  plate  from  dropping  on  the  medium  surface.  Do  not  breathe 
on  the  plate  wThile  filling  it  or  while  inoculating  it.  After 
from  12  to  24  hours'  incubation  of  the  plate  cultures  examine 
them  with  the  naked  eye  and  with  the  hand  glass.  Observe 
the  types  of  colony  present  in  those  parts  of  the  plates  in 
which  the  colonies  are  discrete.  Note  the  colour,  shape  and 
size  of  the  colonies.  Make  film  preparations  if  necessary. 
Subculture  from  each  variety  of  colony  on  to  agar  slopes. 
Incubate  the  sub-cultures  and  the  following  day  pass  them 
through  the  various  media. 

(9)  To  examine  the  sub-cultures. — Note  the  naked-eye 
changes  which   have    occurred   in    each  tube  at  the  end  of 


BACTERIOLOGICAL   METHODS.  155 

24  hours.  Examine  film  preparations  from  one  or  two  of  the 
tubes.  The  complete  changes  will  probably  not  have  taken 
place  in  the  media  after  only  1  clay's  incubation,  but  with 
many  organisms  a  diagnosis  can  be  made  at  this  stage.  Indole 
formation  in  the  broth  tube  may  require  at  least  3  days' 
incubation,  and  so  may  the  production  of  a  green  fluorescence 
in  neutral  red  broth.  The  complete  coagulation  of  milk,  the 
liquefaction  of  gelatin,  and  the  production  of  an  acid  reaction 
in  the  carbohydrate  media  may  require  some  days.  It  is 
advisable,  therefore,  to  postpone  the  indole  test  as  long  as 
possible  and  to  incubate  all  the  tubes  for  from  5  to  7  days 
before  discarding  them. 

Special  methods. — The  preceding  sections  deal  with  the 
routine  bacteriological  examination  of  pus  or  other  body  fluids. 
The  majority  of  the  following  methods  are  special  only  in 
the  sense  that  a  special  organism  is  deliberately  sought  for. 
Nearly  all  the  methods  are  in  common  use.  Further  informa- 
tion as  to  the  organisms  may  be  found  under  the  description 
of  each  in  a  previous  chapter. 

Tuberculosis. —  The  methods  of  demonstrating  the  tubercle 
bacillus  differ  with  the  nature  of  the  suspected  material. 
In  the  case  of  sputum  it  is  advisable  to  collect  a  mixed 
sample  of  the  matter  expectorated  in  the  24  hours. 
When  the  sputum  is  scanty  or  the  tubercle  bacilli  are  few  in 
number,  the  early  morning  sputum  should  be  particularly 
examined.  Quite  young  children  commonly  swallow  the 
sputum,  and  it  may  be  impossible  to  obtain  sufficient  for 
examination.  In  such  cases  the  bacilli  may  be  demonstrated 
in  the  fseces.  Having  obtained  the  sputum,  empty  it  into  a 
wide-mouthed  bottle  provided  with  a  well-fitting  cork.  Add  five 
times  the  volume  of  1  in  20  carbolic  acid.  Shake  thoroughly 
for  about  5  minutes.  Stand  till  the  next  morning.  (The 
object  of  the  carbolic  is  to  separate  the  mucoid  from  the 
purulent  part  of  the  sputum  and  to  a  less  extent  to  clump  the 
tubercle  bacilli.)  After  standing,  pour  off  the  layer  of  mucus, 
which  has  separated  off  at  the  top  of  the  bottle,  together  with 
the  supernatant  fluid,  leaving  the  deposit  of  pus.  Centrifuge 
the  deposit.  Shake  the  sediment  on  to  a  glass  slide  held  in  the 
left  hand.  Take  a  second  glass  slide  in  the  right  hand,  holding 
each  slide  at  its  extremity.  Press  the  top  slide  with  a  to-and- 
fro  movement  several  times  firmly  over  the  under  slide  until 


156  CLINICAL  PATHOLOGY. 

an  even  film  of  sputum  is  spread  over  both  slides.     Wipe  the 
backs  of  the  slides  and  dry  the  films. 

To  stain  the  films  : — Filter  carbol-fuchsin  (full  strength) 
into  a  large-bore  test  tube.  Bring  it  carefully  to  the  boiling- 
point,  constantly  shaking  the  tube  and  keeping  its  mouth 
pointed  away  from  the  face.  Cover  the  slides  with  the  boiling 
stain.  Stain  for  7  minutes,  and  during  that  time  pour  off 
the  stain  twice  and  add  fresh  boiling  stain. 

After  7  minutes  pour  off  the  stain,  dip  the  slide  in  water 
and  then  place  in  25  per  cent,  sulphuric  acid.  (It  is  advisable 
when  dealing  with  two  slides  to  leave  one  staining  in  the 
fuchsin  while  the  other  is  being  decolorised  in  the  acid.) 

When  the  slide  is  decolorised  place  it  in  tap  water.  The 
red  colour  will  probably  return,  in  which  case  replace  in  the 
acid  and  then  return  to  the  water.  Eepeat  the  process  until 
the  pink  colour  fails  to  return  after  immersion  in  water.  .Leave 
in  fresh  water  for  2  minutes. 

Counterstain  in  1  per  cent,  methylene  blue,  diluted  four 
times  with  tap  water,  for  2  minutes.  Wash  in  tap  water.  Dry 
and  mount. 

This  method  is  known  as  the  Ziehl-Neelsen  process. 

In  the  preliminary  staining  with  fuchsin  all  organisms 
and  cells  are  stained  red.  After  the  acid  everything  is 
decolorised  except  the  tubercle  bacillus.  The  final  washing 
in  water  brings  a  brighter  red  colour  into  the  tubercle  bacillus. 
The  counter-stain  renders  other  organisms  and  the  cells  blue. 

The  slides  should  be  examined  under  the  oil  immersion  lens, 
and  at  least  a  quarter  of  an  hour  should  be  spent  on  them 
before  the  tubercle  bacillus  is  pronounced  to  be  absent. 

When  tuberculosis  of  the  lung  is  strongly  suspected  on 
clinical  grounds  and  the  bacilli  cannot  be  demonstrated  by 
this  way,  the  "  antiformin  "  method  may  be  used.  "Anti- 
formin  "  can  be  prepared  by  making  a  stock  solution  of  equal 
parts  of  liquor  sodse  chlorinate  (B.  P.)  and  15  percent,  sodium 
hydrate.  A  20  per  cent,  dilution  of  this  in  tap  water  is  shaken 
with  about  one-fourth  its  bulk  of  sputum  and  left  until  all 
the  gas  has  been  evolved  and  a  fairly  homogeneous  solution 
has  resulted.  The  mixture  is  then  centrifuged  at  a  high 
speed.  Films  are  made  from  the  sediment  and  stained  in  the 
usual  way. 

The  effect  of  the  antiformin  is  to  dissolve  almost  everything 


BACTERIOLOGICAL   METHODS.  157 

in  the  sputum  except  the  tubercle  bacillus,  the  capsule  of  which 
is  not  acted  upon. 

Cultures  on  Dorset's  egg  medium  can  be  made  from  the 
sediment  after  washing  it  free  from  antiformin  with  distilled 
water,  or  animal  inoculations  may  be  carried  out. 

In  the  case  of  urine,  collect  the  deposit  of  the  urine  passed 
during  the  24  hours.  Treat  the  deposit  with  carbolic  acid  and 
prepare  films  in  the  same  manner  as  with  the  sputum.  The 
advantage  of  investigating  the  deposit  obtained  in  this  way 
rather  than  that  from  a  catheter  specimen  is  that  the  bacilli 
are  less  likely  to  be  missed,  since  they  are  not  necessarily 
passed  with  every  sample  of  urine.  The  disadvantage  is  that 
smegma  bacilli  are  likely  to  be  present.  The  smegma  bacillus 
can  be  disregarded  if  the  films  are  passed  through  alcohol. 
In  the  case  of  all  urinary  pus,  therefore,  after  decolorisation 
in  acid,  wash  in  water  and  then  soak  in  methylated  spirit  for 
2  minutes.  Then  wash  in  water  and  counterstain  in  methylene 
blue. 

The  examination  of  urinary  slides  should  be  made  with 
particular  care,  since  numerous  erroneous  diagnoses  of  tubercle 
bacilli  have  been  made.  The  mistake  lies  as  a  rule  in  mis- 
taking, not  the  smegma  bacillus,  but  the  bacilli  of  the  colon 
group,  which  may  be  present  and  stained  red.  The  colon 
bacillus  is  in  no  sense  acid-fast,  but  it  is  not  infrequent  to 
find  that  urinary  crystals,  or  even  large  epithelial  cells,  have 
held  the  fuchsin  stain  in  the  acid,  and  that  the  stain 
diffuses  out  over  a  small  area  in  the  tap-water  stage  and  stains 
the  bacilli  in  that  area  a  bright  red.  Such  bacilli  are  seen  to 
be  lying  on  a  red  background,  and  the  mistake  is  thus  easily 
recognised.  The  slide  must  be  again  decolorised.  The  red 
tubercle  bacilli  must  be  seen  to  lie  on  a  blue  background. 
The  possibility  of  this  error  is  a  very  real  one,  and  a  clump  of 
such  diffusion-stained  bacilli  is  to  be  seen  beautifully  figured, 
and  labelled  as  tubercle  bacilli,  in  a  standard  text-book.  In 
the  case  of  faeces  tubercle  bacilli  are  commonly  very  scanty 
except  when  actual  ulceration  of  the  gut  is  present.  It  is 
advisable  to  take  a  small  quantity  of  the  faeces  and  treat  it 
direct  with  antiformin.  Films  are  made  from  the  centrifuged 
deposit  and  stained  in  the  same  manner  as  for  sputum. 

In  the  case  of  tuberculous  pus,  such  as  may  be  obtained  from  a 
caseous  gland,  a  tuberculous  joint,  etc.,  the  bacilli  are  sometimes 


158  CLINICAL   PATHOLOGY. 

numerous,  but  in  the  great  majority  of  cases  are  extremely 
scanty.  Such  material  is  best  treated  at  once  with  antiformin, 
and  scanty  bacilli  can  frequently  be  demonstrated  in  this  way. 
Antiformin  gives  better  results  with  tuberculous  pus  or  tissues 
than  with  sputum. 

The  detection  of  tubercle  bacilli  in  pleural,  peritoneal,  and 
cerebro- spinal  fluids  is  dealt  with  in  the  section  which  treats 
of  these  exudations. 

The  investigation  of  urine,  faeces,  and  sputum  for  other 
organisms  than  the  tubercle  bacillus  is  considered  under  the 
appropriate  headings. 

The  cultivation  of  tubercle  bacilli  is  rarely  successful  if 
attempted  direct  from  the  human  lesion  owing  to  the  slow 
growth  of  the  bacillus  and  the  frequent  presence  of  other 
readily  growing  organisms ;  nor  is  it  often  necessary  to 
cultivate  the  bacillus,  since  in  the  great  majority  of  cases  it 
can  be  recognised  in  film  preparations.  When  a  culture  of 
the  organism  is  desired,  the  sputum,  urinary  deposit,  or 
caseous  pus  is  first  inoculated  into  a  guinea-pig  and  the 
cultures  are  made  from  the  resulting  lesion  in  the  animal. 
By  the  use  of  antiformin  animal  inoculation  may  be  avoided 
and  the  cultures  can  be  made  on  Dorset's  egg  medium  from 
the  sediment  left  after  centrifuging  the  solution.  The  sedi- 
ment should  be  washed  two  or  three  times  with  sterile  water 
before  inoculating  the  tubes.  It  must  be  confessed  that  the 
antiformin  method  of  treating  sputum  is  often  disappointing, 
and  that  if  the  bacilli  are  not  found  in  the  carbolised  sputum 
they  are  not  likely  to  be  found  or  grown  after  treatment  with 
antiformin. 

The  inoculation  of  animals  is  still  occasionally  necessary 
in  order  to  prove  the  tuberculous  nature  of  a  body  fluid  in 
which  the  bacilli  can  neither  be  found  nor  cultivated,  or  to 
identify  the  exact  nature  of  the  acid-fast  organisms  present. 
The  material  for  inoculation  is  preferably  injected  into  the 
thigh  of  a  guinea-pig,  but  if  the  bulk  is  too  great  for  intra- 
muscular injection,  the  inoculation  is  made  into  the  peritoneal 
cavity.  About  10  days  after  injection  into  the  thigh  the 
inguinal  gland  becomes  palpably  enlarged.  In  from  2  to 
3  weeks  the  spleen  becomes  infected,  and  the  animal 
dies  of  generalised  tuberculosis  from  G  to  8  weeks  after 
inoculation.      The   injections   are   made   with    strict   aseptic 


BACTERIOLOGICAL   METHODS.  159 

precautions,  and  preferably  two  animals  are  inoculated.  After 
killing  the  animal  the  bacilli  are  readily  identified  in  the  more 
advanced  lesions,  which  show  the  typical  gross  and  micro- 
scopical changes  of  tuberculosis. 

Diphtheria. — The  taking  of  swab  and  culture  for  the 
identification  of  the  diphtheria  bacillus  and  the  differentiation 
of  this  organism  from  similar  bacteria  have  been  described 
under  the  heading  of  the  diphtheria  bacillus. 

The  examination  of  the  swab  and  culture  is  conducted  as 
follows  : — Twelve  hours  incubation  at  37°  C.  is  sufficient  to 
obtain  a  growth  of  the  organism.  After  incubation  examine 
the  blood  serum  tube  with  the  naked  eye  and  with  a  hand- 
glass. 

Clean  two  slides  and  place  a  drop  of  water  in  the  centre  of 
each.  On  one  slide  make  a  thin  film  from  the  most  suspicious 
colonies  on  the  serum  slope.  On  the  other  slide  make  as  thick 
a  film  as  possible  by  rubbing  the  swab  in  the  drop  of  water. 
Dry  and  fix  the  slides. 

Pour  on  Lofiier's  methylene  blue  and  leave  it  for  3  minutes. 

Wash  in  tap  water.     Blot  dry  and  mount. 

Lofner's  methylene  blue  consists  of  an  alcoholic  solution  of 
the  dye  with  potassium  hydrate  added.  The  beading  of  the 
bacillus  is  more  satisfactorily  displayed  by  means  of  this 
stain  than  with  the  ordinary  dyes  such  as  carbol-thionin. 

The  identification  of  the  bacillus  on  morphological  grounds 
is  a  matter  requiring  practice,  and  no  real  additional  informa- 
tion is  gained  by  more  elaborate  staining  methods,  although 
the  student  is  advised  to  confirm  his  opinion  with  a  Gram 
preparation. 

Neisser's  method  of  staining  is  still  in  common  use,  and 
those  who  prefer  brown  bacilli  with  blue  dots  to  alternating 
pale  and  dark  blue  organisms  should  use  this  method.  The 
differential  staining  is  no  real  criterion  of  the  virulent  as 
opposed  to  the  diphtheroid  organisms,  but  is  a  help  to  those 
unacquainted  with  the  morphology  of  this  group  of  bacilli. 
The  method  of  staining  is  as  follows  : — 

Make  a  mixture  of  2  parts  of  a  solution  composed  of 
Methylene  blue  powder,  1  gramme  ; 
Absolute  alcohol,  50  c.c. ; 
Glacial  acetic  acid,  50  c.c. ; 
Distilled  water,  1,000  c.c. ; 


160  CLINICAL  PATHOLOGY. 

and  1  part  of  the  solution 

Crystal  violet,  1  gramme  ; 
Absolute  alcohol,  10  c.c. ; 
Distilled  water,  300  c.c. 
Stain  in  this  mixture   for   2    seconds.      Wash  rapidly  in 
water. 

Counterstain  for  3  seconds  in  the  following  solution  : — 
Cresoidin,  1  gramme 
(dissolved  in  300  c.c.  of  warm  water  and  filtered). 
Wash  in  water.     Dry  and  mount. 

The  body  of  the  bacillus  is  stained  brown  and  the  granules 
blue. 

Syphilis. — The  method  of  obtaining  the  material  for 
examination  has  been  described  in  the  section  on  the 
SpirocJiceta  pallida.  The  fluid  is  examined  in  the  following 
ways : — 

(a)  The  Indian  ink  method.  The  most  suitable  variety  of 
Indian  ink  is  that  known  as  "  chin  chin  liquid  pearl."  The 
bottle  must  not  be  shaken. 

Place  at  one  end  of  a  clean  slide  a  small  drop  of 
the  ink. 

Take  a  platinum  loop  of  the  secretion  and  thoroughly  mix 
it  with  the  ink.  With  a  second  slide  make  a  thin  film  of 
the  mixture  in  the  same  manner  as  if  making  a  blood 
film. 

When  dry  examine  direct  with  the  oil  immersion  lens, 
using  artificial  light.  The  spirochetes  are  seen  as  white 
refractile  threads  on  a  brownish-black  background. 

(b)  By  Giemsa's  stain.  Make  on  clean  slides  two  or 
three  films  of  the  secretion  with  a  platinum  loop. 

When  dry  cover  with  absolute  alcohol  and  leave  for  15 
minutes. 

Make  up  from  the  stock  Giemsa's  stain  a  mixture  of 
10  c.c.  of  distilled  water  and  14  drops  of  the  stain. 

Pour  off  the  alcohol  and  cover  with  the  stain  for  45 
minutes. 

Wash  in  a  rapid  stream  of  distilled  water. 

Dry  and  examine  with  a  -^-inch  objective. 

The  Spirochceta  pallida  stains  a  rose-pink  colour,  other 
spirochetes  and  bacteria  blue.  The  difference  in  shade 
between  the  syphilitic  and  other  spirochetes    is   frequently 


BACTERIOLOGICAL   METHODS.  161 

far  from  obvious,  and  greater  reliance  is  to  be  placed  on  the 
morphological  points  of  distinction. 

(c)  The  dark  ground  illumination  (the  ultra-microscope). 
A  special  condenser  (the  paraboloid  condenser)  is  required  for 
this  method  of  investigation.  It  is  fitted  into  the  collar  pre- 
pared for  the  ordinary  condenser.  A  "  stop  "  is  also  used 
which  fits  into  the  inside  of  the  j^-inch  objective.  Special 
thin  slides  and  cover-glasses  should  also  be  obtained.  The 
illuminant  must  be  a  powerful  one,  and  should  either  be  a 
small  arc  or  a  Nernst  lamp.  The  microscope  should  be 
vertical. 

A  drop  of  the  fluid  is  placed  on  the  centre  of  the  slide,  and 
if  only  a  small  quantity  is  available  saline  should  be  added. 

The  cover-glass  is  carefully  let  down  on  to  the  slide  so  that 
all  aiu  bubbles  may  be  avoided. 

A  drop  of  cedar  oil  is  placed  on  the  upper  surface  of  the 
condenser  and  another  on  the  under  surface  of  the  slide.  The 
condenser  is  racked  up  until  the  two  drops  coalesce. 

A  third  drop  of  oil  is  placed  on  the  upper  surface  of  the 
cover-glass,  and  the  objective  is  focussed  after  first  directing 
all  the  available  light  through  the  preparation. 

Instead  of  an  immersion  lens  the  J-inch  objective  and  a 
high  eyepiece  may  be  substituted. 

The  spirochetes  show  as  shining  white  refractile  bodies  on 
a  black  background. 

A  great  advantage  of  this  method  is  that  the  motility  of  the 
organisms  can  be  studied. 

The  bacterial  investigation  of  viscera. 

It  is  occasionally  necessary  to  investigate  the  bacterial 
content  of  a  closed  viscus,  such  as  a  cyst  or  a  pyosalpinx. 
The  procedure  is  as  follows  : — 

Place  the  specimen  on  a  clean  plate.  Heat  a  metal  spatula 
to  red  heat  in  the  flame  and  smear  it  firmly  over  the  surface  of 
the  specimen.  With  a  sterile  knife  cut  through  the  seared 
surface  into  the  centre  of  the  specimen.  Hold  the  edges  of 
the  cut  apart  with  a  pair  of  sterile  forceps.  With  a  platinum 
loop  make  films  and  cultures  from  the  pus  or  from  the  fluid 
in  the  viscus. 

The  bacteriological  examination  of  viscera  post  mortem  is 
conducted  in  the  same  manner.  In  cases  of  general  infection, 
and  particularly  in  typhoid  fever,  the  causative  organism  may 

p.  11 


162  CLINICAL   PATHOLOGY. 

be  obtained  in  pure  culture  from  the  spleen  juice.  Owing  to 
the  rapid  emigration  of  organisms  from  the  intestinal  tract 
into  the  viscera  at  the  time  of  death  post-mortem  bacterial 
findings  should  always  be  accepted  with  reserve. 

Anaerobic  cultures. 

By  an  anaerobic  culture  is  meant  the  incubation  of  an 
organism  in  an  atmosphere  free  from  oxygen.  The  simplest 
method  of  obtaining  such  an  atmosphere  is  by  means  of 
Buchner's  tubes.  These  tubes  consist  of  long,  wide-bore  test 
tubes  constricted  a  few  inches  from  the  bottom  in  such  a  way 
that  an  ordinary  test  tube  passed  into  the  Buchner's  tube 
can  rest  upon  the  constriction.  The  tube  is  supplied  with  a 
well-fitting  rubber  cork. 

To  put  up  the  anaerobic  culture : — 

Shake  pyrogallic  acid  powder  into  the  Buchner's  tube  so  as 
to  fill  the  space  between  the  bottom  of  the  tube  and  the  con- 
striction. Pour  in  15  per  cent,  caustic  potash  to  the  level 
of  the  constriction.  Kapidly  slide  in  (do  not  drop  in)  the 
inoculated  culture  tube,  with  wool  plug  in  place. 

Bapidly  fix  in  the  rubber  cork  of  the  Buchner's  tube. 

Plaster  round  the  junction  of  the  cork  and  the  tube  with 
soft  paraffin. 

The  alkaline  pyrogallic  solution  absorbs  the  oxygen  from 
the  interior  of  the  Buchner's  tube  and  of  the  culture  tube. 

Special  staining  processes. 

Capsules. — Cover  the  films  for  3  minutes  with  1  per  cent, 
acetic  acid. 

Drain  off  the  acetic  acid  and  dry. 

Stain  for  10  seconds  in  aniline  gentian  violet  (see  under 
Gram's  stain). 

Wash  in  water  and  examine  in  water. 

If  the  capsule  is  stained  too  deeply  wash  in  1  per  cent, 
acetic  acid. 

Dry  and  mount. 

This  method  of  staining  is  not  infrequently  unsuccessful  and 
is  rarely  called  for. 

Spores. — Fix  the  film  after  drying  by  passing  many  times 
through  the  flame. 

Stain  with  boiling  carbol-fuchsin,  giving  several  changes, 
for  10  minutes. 

Dip  in  acid  alcohol  (99  c.c.  alcohol,  1  c.c.  HC1). 


BACTERIOLOGICAL   METHODS.  163 

Blot  dry. 

Stain  in  dilute  methylene  blue  2  minutes. 

Wash  in  water.     Dry.     Mount. 

The  spores  are  red  and  the  bacilli  blue.  The  difficulty  with 
the  process  lies  in  the  differentiation  with  acid  alcohol. 
With  the  majority  of  spores  it  is  difficult  to  get  the  fuchsin 
to  penetrate  them  sufficiently  to  allow  more  than  the 
most  rapid  dipping  in  the  spirit.  Successful  preparations 
have  quite  a  striking  appearance,  but  the  student  should  be 
able  to  recognise  with  certainty  spores  in  ordinary  carbol- 
thionin  preparations  and  to  distinguish  them  from  beading  of 
the  bacillus  by  their  regular  shape  and  clean-cut  outline. 

Flagella. — Film  preparations  are  made  from  a  growth  on 
a  solid  medium. 

In  making  the  films  care  is  taken  to  avoid  picking  up  any 
of   the   medium   and   to  spread  the   bacteria  as  thinly  and 
evenly  as  possible.     Prepare  the  following  mordant : — 
Tannin,  2  grammes. 
Water,  20  c.c. 

Ferrous  sulphate  solution  of  1 : 2  strength,  4  c.c. 
Saturated  alcoholic  solution  of  fuchsin,  1  c.c. 

Pour  mordant  over  film  and  heat  without  boiling  1  minute. 

Wash  in  water.     Stain  with  carbol-fuchsin. 

Wash  in  water.     Dry.     Mount. 

This  method  is  described  as  the  most  simple  of  the  flagella 
staining  processes.  There  are  so  many  cultural  and  other 
methods  of  differentiating  the  flagellated  bacteria  that  the 
demonstration  of  the  flagella  is  rarely  necessary. 


11—2 


CHAPTEE    XII. 

vaccines anti-sera. 

Vaccines. 

A  vaccine  is  a  sterile  standardised  suspension  of  dead 
organisms  in  a  neutral  fluid.  It  is  given  hypodermically  in 
doses  graduated  according  to  the  actual  number  of  bacteria 
present  in  each  dose.  The  object  of  vaccine  treatment  is  to 
raise  the  resistance  of  the  individual  to  the  organism  with 
which  he  is  infected  by  carefully  graded  and  spaced  doses  of 
dead  organisms.  The  toxins  contained  in  the  dead  bacteria 
are  liberated  in  the  tissues,  passed  into  the  circulation,  and 
thus  lead  to  an  over-production  of  antibodies  which  can 
be  used  to  combat  the  living  organisms  of  the  disease. 
Less  frequently  vaccines  are  given  to  a  healthy  individual 
with  the  view  of  rendering  him  immune  to  a  possible  infection. 
The  immunity  induced  by  vaccines  is  an  artificial  acquired 
immunity.  It  is  established  comparatively  slowly,  over  a 
period  of  weeks,  and  lasts  a  comparatively  long  time. 

The  treatment  of  a  patient  with  vaccines  necessitates  a 
co-operation  between  the  pathologist  and  the  clinician,  since 
it  is  insufficient  in  the  great  majority  of  cases  to  raise  the 
immunity  of  a  patient  to  a  given  organism.  The  local  and 
general  treatment  of  the  disease  must  also  be  undertaken 
on  the  usual  lines.  The  cure  of  a  case  of  gonorrhceal 
arthritis,  for  example,  may  be  greatly  expedited  by  means  of 
a  vaccine,  but  the  vaccine  may  be  almost  useless  in  the 
presence  of  an  untreated  stricture  or  a  posterior  urethritis. 
If  a  local  source  of  infection  is  left  unrelieved  the  organisms 
present  in  it  continue  to  multiply  and  to  aggravate  the  general 
condition.  A  patient  with  puerperal  septicaemia  may  die  if  only 
the  uterus  is  drained,  or  if  a  vaccine  is  given  without  cleansing 
the  uterus.  A  combination  of  both  procedures  has  undoubtedly 
saved  many  lives.  In  such  a  condition  as  infective  endocarditis, 
in  which  the  source  of  infection  cannot  be  dealt  with,  vaccine 
treatment  is  practically  useless. 


VACCINES— ANTI-SERA.  165 

There  is  no  doubt  that  vaccines  have  been  given  in  the  past 
in  a  wild  and  extravagant  manner,  and  the  field  of  vaccine 
therapy  is  now  becoming  more  restricted.  The  following  are 
among  the  conditions  for  which  vaccines  may  reasonably  be 
given : — 

Chronic  local  infections,  such  as  acne,  recurrent  boils  and 
local  abscesses,  of  which  the  causative  organism  is  certainly 
known,  and  is  usually  a  staphylococcus,  or,  in  the  case  of 
uncomplicated  acne,  the  acne  bacillus. 

Acute  streptococcal  infections,  particularly  those  in  which 
the  organisms  have  been  isolated  from  the  general  circulation, 
and  the  local  source  of  infection  is  accessible. 

Gonorrheal  infections,  particularly  when  chronic,  less 
certainly  in  the  stage  of  acute  urethritis. 

In  the  following  conditions  the  use  of  vaccines  is  more 
problematical,  but  worthy  of  trial  in  individual  cases:  — 

Pyorrhoea  alveolaris  and  its  probable  complications. 

Chronic  infection  of  the  urinary  tract  with  the  B.  coli. 

Local  abscess,  such  as  a  bronchiectatic  cavity,  in  which  the 
organism  isolated  is  the  probable  source  of  infection. 

Vaccines  should  not  be  given  merely  for  the  sake  of  doing- 
something.  For  example,  a  case  of  rheumatoid  arthritis 
obtained  no  benefit  from  medical  treatment.  A  catheter 
specimen  of  urine  was  taken  and  found  sterile.  A  blood 
culture  was  sterile.  Cultures  from  the  faeces  yielded  a  growth 
of  the  typical  colon  bacillus.  Cultures  were  finally  taken 
from  the  perfectly  healthy  gums,  a  coccus  was  grown,  and  a 
vaccine  made  from  it.  Such  a  proceeding  is  quite  futile,  if 
harmless. 

Stock  vaccines  should  not  be  given  on  the  chance  of  hitting 
off  the  correct  species  of  organism,  when  it  is  possible  to  make 
a  proper  bacteriological  investigation  and  to  grow  the  causative 
organism. 

While  it  is  probable  that  the  majority  of  vaccines  do  no  harm, 
it  must  be  recognised  that  in  some  cases  the  injection  of  dead 
organisms  is  positively  dangerous.  Some  cases  of  infective 
endocarditis  are  undoubtedly  made  worse  by  vaccine  treat- 
ment, and  the  subjects  of  grave  anaemia  may  succumb  to  large 
doses  of  an  organism  with  which  they  are  not  actively  infected. 
For  example,  a  patient  with  pernicious  anaemia  of  some 
years'  standing  who  was  in  comparatively  good  condition  was 


166  CLINICAL  PATHOLOGY. 

given  a  vaccine  prepared  from  a  streptococcus  isolated  from 
the  mouth.  He  reacted  strongly  to  the  vaccine,  which  was 
repeated,  and  he  was  dead  within  a  fortnight. 

The  above  are  only  the  merest  indications  of  when  or  when 
not  to  treat  a  patient  with  vaccines.  Each  case  must  be  judged 
on  its  merits,  but  it  can  be  stated  broadly  that  the  cases  most 
benefited  are  some  chronic  septic  conditions  and  general 
streptococcal  infections.  The  dosage  of  a  vaccine  necessarily 
varies  with  the  age,  size,  and  general  condition  of  the  patient, 
as  well  as  with  the  species  of  the  organism  injected.  The 
variations  as  to  age  and  condition  are  similar  to  those  employed 
in  any  form  of  treatment.  The  dosage,  according  to  species 
of  organism,  varies  with  the  comparative  virulence  of  the 
organism.  In  the  cases  of  streptococci,  pneumococci,  and  gono- 
cocci  small  initial  doses  of  from  5  to  10  million  cocci  are  given 
as  a  general  rule.  In  the  cases  of  staphylococci  and  colon  bacilli 
doses  of  from  50  to  100  million  organisms  are  given.  Double 
these  amounts  may  usually  be  injected  in  the  second  dose. 

The  vaccine  may  be  given  as  soon  as  it  is  prepared,  but  if 
a  surgical  operation  has  recently  been  performed,  and  there  is 
reason  to  suppose  that  the  patient  has  absorbed  a  material 
dose  of  toxin  from  the  operation  area,  the  giving  of  the  initial 
dose  should  be  postponed.  If  there  is  urgency  in  giving  the 
vaccine,  a  probable  idea  of  the  causative  organism  justifies  the 
use  of  a  preliminary  dose  of  a  stock  culture  of  that  organism. 
Nor  is  there  any  grave  objection  to  treatment  by  stock  vaccines 
if  the  proper  organism  is  known,  although  the  balance  of 
evidence  seems  to  be  that  patients  are  more  likely  to  improve 
on  an  autogenous  vaccine — that  is,  a  vaccine  prejDared  with  the 
organisms  obtained  from  the  individual  case — than  on  a  vaccine 
derived  from  some  other  source.  The  time  allowed  to  elapse 
between  the  doses  is  as  a  rule  from  5  to  10  days.  There  is 
nothing  to  be  gained  by  increasing  the  size  of  the  dose  to 
much  more  than  double  the  initial  dose,  nor  by  markedly 
diminishing  the  intervals  between  the  doses. 

The  immediate  effect  of  giving  a  vaccine  is  apparently  to 
diminish  the  resistance  of  the  individual  to  the  infection. 
The  diminished  resistance  is  followed  by  a  considerable  rise  in 
the  immune  bodies.  The  rise  is  succeeded  by  a  fall  to  a  point 
somewhat  above  the  resistance  before  inoculation.  The 
clinical  signs  vary  with  the  rise  in  immunity,  the  temperature 


VACCINES— ANTI-SEEA.  167 

falling  and  the  condition  improving.  In  most  cases  the 
patient  experiences  little  or  no  discomfort  after  the  vaccine  is 
given,  but  in  a  certain  percentage  of  cases  a  definite  reaction 
follows.  The  reaction  may  be  local,  focal,  or  general,  or  all 
three.  The  local  reaction  consists  of  pain,  redness  and  even 
oedema,  at  the  site  of  injection.  The  general  reaction  com- 
prises a  rise  of  temperature,  with  headache,  malaise  and 
sometimes  sickness.  A  focal  reaction  consists  in  an  aggrava- 
tion of  the  local  condition.  A  marked  reaction  is  evidence  of 
an  overdose  for  the  individual. 

The  control  of  vaccines  by  frequent  estimations  of  the 
opsonic  index  has  been  already  referred  to  (Chapter  IV.).  The 
method  has  been  largely  abandoned,  and  a  sufficient  guide  to 
the  effect  of  the  treatment  is  afforded  by  the  temperature 
chart,  combined  with  clinical  observation  of  the  patient. 

Certain  modifications  of  vaccine  treatment  may  be  men- 
tioned only,  since  they  have  yet  to  be  put  into  practice  on  any 
large  scale.  Treatment  by  endotoxins  has  been  attempted  for 
several  diseases,  and  in  particular  for  typhoid  fever.  The 
endotoxins  are  obtained  by  grinding  up  the  bacilli  in  such  a 
way  as  to  actually  express  from  them  the  intracellular  toxins. 
The  sensitised  vaccine  of  Besredka  consists  of  a  vaccine  which 
has  been  brought  into  contact  with  the  specific  antiserum  so 
that  the  organisms  in  the  vaccine  are  combined  with  the  anti- 
body in  the  serum.  It  is  claimed  that  such  a  vaccine  is  non- 
toxic, leads  to  no  preliminary  lowering  of  the  immunity,  raises 
the  immunity  very  rapidly,  and  produces  an  immunity  which 
lasts  a  considerable  time. 

Tuberculin  is  given  in  many  different  forms,  and  only  one 
variety,  tuberculin  B  E,  is  a  genuine  vaccine,  in  the  sense  that 
it  consists  of  a  known  weight  of  tubercle  bacilli  emulsified 
with  a  mixture  of  glycerine  and  water.  The  old  tuberculin 
consists  of  exotoxins  mainly,  and  is  obtained  by  evaporating 
down  a  4  to  5  weeks'  old  culture  of  bacilli  to  one-tenth 
its  bulk  at  a  comparatively  low  temperature,  filtering  it  and 
using  the  filtrate.  Tuberculin  T  R  is  composed  of  endotoxins, 
and  1  c.c.  of  the  T  B  contains  the  bacterial  matter  insoluble  in 
water  derived  from  10  mg.  of  tubercle  bacilli  which  have  been 
repeatedly  washed  in  water  and  centrifuged.  The  S  B  E  con- 
sists of  sensitised  tubercle  bacilli  obtained  by  mixing  the 
bacilli  with  antituberculous  serum. 


168  CLINICAL   PATHOLOGY. 

The  preparation  of  the  tuberculins  is  largely  left  to  the 
manufacturing  chemist. 

The  dosage  of  the  tuberculins  varies  with  the  nature  of  the 
case,  and  is  reckoned,  so  far  as  possible,  from  the  weight  of 
original  solid  substance  employed  to  make  the  extract,  or  from 
the  original  solution. 

In  the  case  of  B  E,  for  example,  the  preliminary  dose  is  a 
dilution  of  the  original  solution,  and  is  commonly  "001  of  a 
cubic  millimetre.  This  may  be  given  as  the  initial  dose  of 
any  tuberculin.  The  dose  is  increased  gradually  and  in  such 
a  way  as  the  following :— '001,  -002,  '003,  -004,  -006,  -008, 
•010,  *015,  "02,  -03,  etc.  The  maximum  dose  to  be  aimed  at 
is  100  c.mm.,  or  ^6  c.c.  of  the  original  solution. 

The  intervals  between  the  doses  should  be  3  days  for  the 
small  doses,  and  from  1  to  3  weeks  when  the  higher  doses 
are  reached. 

The  rate  of  increase  necessarily  depends  upon  the  reaction 
and  progress  of  the  patient. 

The  value  of  tuberculin  treatment  cannot  even  yet  be  con- 
sidered as  proved.  There  is  no  doubt  that  in  careless  hands 
much  harm  can  be  done  with  tuberculin.  On  the  other  hand, 
many  careful  observers  have  claimed  satisfactory  results  from 
its  use. 

The  method  of  preparing  vaccines. — The  following  are 
the  various  stages  requisite  for  the  preparation  of  an  auto- 
genous vaccine  : — 

Stage  1.  The  culture. — Cultures  are  taken  from  the 
patient  in  the  usual  way,  and  film  preparations  of  the  lesions 
are  also  examined  if  possible.  A  sub-culture  on  agar  (or,  if 
necessary  for  the  growth  of  the  organism,  on  serum  agar)  is 
made  and  incubated  for  24  hours.  A  second  sub-culture  should 
be  put  up  at  the  same  time  for  the  purpose  of  full  cultural 
investigation.  If  the  organism  is  in  pure  culture  there  is  no 
difficulty  at  this  stage,  and  the  original  culture  on  agar  may 
be  used  if  there  is  an}'  urgency  about  the  preparation  of  the 
vaccine.  If  more  than  one  organism  is  present,  it  is  advisable 
to  make  the  vaccine  from  the  more  virulent  bacterium,  par- 
ticularly if  it  predominates  in  the  film  preparation  and  is  most 
likely  from  the  nature  of  the  case  to  be  the  primary  cause  of 
the  lesion.  For  example,  if  a  Staphylococcus  alius  and  a 
Streptococcus  pyogenes  are  obtained  from  an  acute  cellulitis, 


VACCINES— ANTI-SERA.  169 

the  vaccine  should  be  made  from  the  streptococcus.  In  cases 
of  doubt,  or  when  there  is  reason  to  suppose  that  a  combination 
of  organisms  is  responsible  for  the  condition,  vaccines  can  be 
prepared  from  two  or  more  organisms  and  subsequently  mixed 
in  whatever  proportions  are  considered  desirable. 

Stage  2.  The  suspension. — To  the  24  hours'  old  sub-cul- 
ture on  an  agar  slope  add  sterile  1  per  cent,  salt  solution.  The 
amount  of  saline  to  be  added  depends  upon  the  thickness  of 
the  growth.  A  moderately  turbid  suspension  of  bacteria  in 
saline  is  to  be  aimed  at.  Wash  off  the  growth  as  far  as 
possible  into  the  saline  by  shaking  the  tube  and  pour  the 
bacterial  suspension  into  a  sterile  tube. 

Stage  3.  The  standardisation.— Make  ready  a  Wright's 
opsonic  pipette,  2  clean  slides  such  as  are  used  for  blood 
work,  clean  swabs  and  ether,  a  surgical  needle,  and  a  bowl  of 
1  in  20  carbolic. 

Shake  the  saline  suspension  very  thoroughly  for  several 
minutes  and  hand  it  to  an  assistant  to  continue  the  shaking. 
Make  a  pencil  mark  about  half  an  inch  from  the  end  of  the 
Wright's  pipette.  Cleanse  the  lobe  of  the  assistant's  ear  care- 
fully with  ether  and  prick  it  with  the  needle.  Without  apj)ly- 
ing  pressure  to  the  lobe  draw  up  a  volume  of  blood.  Wipe 
the  end  of  the  pipette.  Take  the  saline  suspension  from  the 
assistant  (the  wool  plug  having  been  withdrawn)  and  draw  up 
a  volume  of  the  suspension.  Mix  saline  and  blood  thoroughly 
on  a  glass  slide.  Draw  up  mixture  into  tube.  Blow  out 
mixture  on  to  a  clean  slide  and  make  a  thin  film  as  in 
preparing  a  blood  film.  Place  all  materials  which  have  been 
in  contact  with  the  organisms  in  the  carbolic  bowl. 

Stain  the  film  with  Leishman's  stain. 

Fit  a  square  aperture,  such  as  is  used  for  the  enumeration 
of  leucocytes,  into  the  eye-piece  of  the  microscope.  Use  the 
-j^-inch  objective.  Choose  a  thin  and  evenly  spread  part  of 
the  film  and  count  500  red  cells  together  with  all  the 
organisms  lying  among  them. 

Enumerate  the  red  cells  per  cubic  millimetre  of  the 
assistant's  blood. 

Sufficient  data  have  been  obtained  to  arrive  at  the 
number  of  organisms  per  cubic  centimetre  of  the  saline 
suspension. 

The   number  of   organisms   counted  among  500  red  cells 


170  CLINICAL   PATHOLOGY. 

multiplied  by  twice  the  number  of  red  cells  per  c.mm.  gives 
the  number  of  organisms  per  cubic  centimetre  of  the 
suspension. 

The  method  given  here  is  perhaps  the  simplest  in  common 
use.  It  is  admittedly  open  to  criticism,  but  is  sufficiently 
accurate  for  all  practical  purposes. 

The  preparation  of  the  film  from  the  mixture  of  blood  and 
saline  must  be  prepared  before  heating  the  suspension,  since 
some  organisms  lose  their  staining  properties  on  being  sub- 
jected to  heat.  The  actual  enumeration  of  the  film,  however, 
is  preferably  postponed  until  after  stage  5. 

Stage  4.  The  sterilisation. — Prepare  a  deep  water  bath 
or  a  saucepan  capable  of  holding  a  considerable  bulk  of  water. 
Fill  almost  to  the  brim  with  water  at  60°  C.  Place  over  a 
Bunsen  flame  and  lower  the  flame  considerably.  Leave  a 
thermometer  in  the  water.  By  adjusting  the  size  of  the 
flame  the  temperature  can  be  maintained  at  60°  C.  without 
appreciable  variation.  When  the  temperature  has  become 
constant,  fit  a  rubber  cap  over  the  mouth  and  cotton-wool 
plug  of  the  vaccine  tube  and  immerse  the  tube  in  the  water 
almost  to  the  level  of  the  rubber  cap.  The  tube  can  be 
conveniently  maintained  in  position  by  adjusting  two  long- 
handled  test-tube  holders.  Keep  the  tube  at  60°  C.  for  1 
hour,  observing  the  reading  of  the  thermometer  from  time  to 
time.  At  the  end  of  1  hour  remove  the  tube.  The  actual 
temperature  necessary  to  kill  the  organism  varies  with  the 
species.  The  majority  of  organisms  growing  in  pin-point 
colonies,  such  as  gonococci,  streptococci  and  pneumococci, 
are  killed  by  exposure  to  60°  C.  in  from  30  to  45  minutes : 
organisms  growing  in  coarser  colonies,  such  as  staphylococci 
and  colon  bacilli,  require  a  temperature  of  at  least  65°  C.  for 
an  hour,  and  with  some  strains  it  may  be  necessary  to  raise 
the  temperature  higher  still. 

Stage  5.  The  proof  of  sterilisation.  —  Incubate  the 
vaccine  tube  for  24  hours.  At  the  end  of  this  time  sub- 
culture from  the  vaccine  on  to  an  agar  slope  and  incubate 
the  sub-culture  for  a  further  24  hours. 

If  the  sub-culture  remains  sterile,  the  vaccine  may  be 
considered  sterile.  The  slide  prepared  in  stage  3  may  then 
be  enumerated  and  stage  6  proceeded  with. 

Stage  6.  The  dilution. — Prepare  sterile  measure  glasses, 


VACCINES— ANTI-SERA..  171 

a  small  sterile  bottle  with  a  well-fitting  ground  glass  stopper, 
sterile  normal  saline,  and  a  bottle  of  lysol. 

Calculate  the  convenient  dilution  of  the  bacterial  suspension. 
For  example,  if  a  streptococcal  suspension  has  been  found  to 
contain  500  million  cocci  per  cubic  centimetre,  and  the  initial 
dose  wished  for  is  10  million,  it  is  convenient  to  add  4  volumes 
of  saline  to  1  volume  of  the  suspension,  thus  reducing  the 
strength  of  the  suspension  to  100  million  cocci  per  c.c.  The 
initial  dose  will  then  be  contained  in  one-tenth  of  a  cubic 
centimetre. 

Sufficient  lysol  should  be  added  to  the  saline  to  provide  in 
the  final  mixture  a  lysol  solution  of  a  strength  of  0*25  per 
cent.  The  addition  of  the  lysol  safeguards  the  vaccine  from 
subsequent  contamination. 

The  mixture  is  finally  placed  in  a  sterile  bottle  and  kept  in 
a  dark,  cool  place.  The  bottle  should  be  marked  with  the 
name  of  the  patient,  the  nature  of  the  organism,  the  strength 
and  the  date. 

It  is  advisable  to  use  no  vaccines  of  a  greater  age  than 
3  months. 

The  above  steps  are  those  preferably  observed.  It  will  be 
noticed,  however,  that  2  days  are  required  for  the  sub-culture 
and  2  more  for  the  proof  of  sterilisation,  so  that  if  every- 
thing goes  well  4  days  are  required  for  the  preparation  of 
the  vaccine. 

In  cases  of  urgency  a  stock  vaccine  may  be  given  at  once. 

In  acute  streptococcal  infections  it  is  possible  to  prepare 
a  vaccine  in  24  hours.  The  original  culture  on  agar  is  taken 
and,  if  pure,  the  suspension  is  made  and  heated  to  65°  C.  for 
1  hour  and  for  10  minutes  at  70°  C.  It  is  then  presumed 
sterile,  and  is  diluted  and  injected.  There  is  in  practice  no 
real  risk  in  this  procedure. 

To  give  the  vaccine. — A  1  c.c.  hypodermic  glass  syringe 
graduated  in  one-tenths  of  a  cubic  centimetre  is  required.  The 
syringe  is  conveniently  sterilised  with  boiling  methylated  spirit. 
A  small  quantity  of  the  spirit  is  placed  in  a  beaker  and  brought 
to  the  boil  on  a  sand-bath  over  a  Bunsen  burner.  The  flame  is 
turned  out  when  the  spirit  is  boiling  briskly,  and  the  syringe 
with  needle  attached  is  washed  in  and  out  several  times  with 
the  boiling  spirit.  The  vaccine  bottle  must  be  thoroughly 
shaken  before  drawing  up  into  the  empty  syringe  the  required 


172  CLINICAL   PATHOLOGY. 

volume  of  the  vaccine.  The  injection  is  preferably  made 
subcutaneously  into  the  forearm  after  cleansing  the  skin  with 
ether.  The  puncture  should  subsequently  be  sealed  with  a 
drop  of  collodion. 

The  prophylactic  use  of  vaccines. — Vaccines  may  be 
given  with  the  object  of  preventing  disease.  Almost  the 
only  preventive  vaccination  of  this  kind  commonly  performed 
in  this  country  is  that  for  typhoid  fever.  Vaccination  against 
small-pox  is  of  a  somewhat  different  nature,  since  the  individual 
is  protected  by  an  artificial  attack  of  the  modified  disease. 
Preventive  inoculation  is  also  practised  against  plague,  cholera, 
and  other  diseases.  The  effects  of  inoculation  against  typhoid 
are  to  markedly  lessen  the  risk  of  infection  and  to  modify  the 
virulence  of  the  attack  if  it  does  occur.  There  is  sufficient 
statistical  evidence  that  a  considerably  smaller  percentage  of 
inoculated  persons  become  attacked  after  exposure  to  infection 
than  of  untreated  persons,  and  that  the  incidence  mortality 
rate  among  the  inoculated  is  very  low.  The  effect  of  the 
inoculation  gradually  wears  off  and  the  comparative  immunity 
does  not  appear  to  last  longer  than  from  one  to  two  years. 
Typhoid  fever  is  not  sufficiently  prevalent  in  Britain  to  call 
for  preventive  inoculation ,  but  inoculation  should  be  advised  for 
those  proceeding  to  countries  where  the  disease  is  rife.  While 
abroad  all  the  usual  precautions  against  infection  should  be 
observed  and  re-inoculation  should  be  performed,  if  practicable, 
within  18  months. 

The  vaccine  should  be  given  a  short  time  before  the  typhoid 
district  is  reached.  Two  doses  are  given  with  a  10-day 
interval  between  them.  Comparatively  large  doses  are 
commonly  given,  often  from  500  to  800  million  organisms, 
and  it  is  reasonable  to  give  a  combined  vaccine  prepared  from 
typhoid  and  paratyphoid  bacilli.  The  inoculations  in  a  con- 
siderable percentage  of  cases  are  followed  by  a  definite  local 
reaction  and  often  by  slight  pyrexia  and  malaise.  The  patient's 
serum  after  inoculation  should  show  well-marked  agglutinating 
action  upon  the  bacilli. 

The  diagnostic  use  of  vaccines. — Vaccines  or  bacterial 
extracts  may  be  made  use  of  for  purposes  of  diagnosis.  The 
reaction  depends  upon  the  fact  that  persons  infected  by  a 
given  organism  are  abnormally  susceptible  to  the  toxins  of 
that  organism;  consequently  the  introduction  of  the  toxins 


VACCINES— ANTI-SERA.  173 

locally  leads  to  an  excessive  local  reaction  and,  if  the  dose  is 
sufficient,  to  a  general  reaction  also.  Necessarily  the  dose 
given  must  be  regulated,  since  an  excessive  dose  might  lead 
to  severe  reaction  in  normal  people  or  to  an  excessive  reaction 
in  infected  persons. 

The  most  widely  used  diagnostic  extract  of  this  nature  is 
tuberculin,  and  it  is  given  by  several  methods. 

The  ophthalmic  reaction  of  Calmette  consists  in  dropping 
a  tuberculin  solution  into  one  eye.  Tuberculous  patients 
develop  a  conjunctivitis,  while  normal  persons  are  unaffected. 
A  grave  objection  to  this  method  lies  in  the  comparative 
frequency  with  which  a  severe  and  intractable  conjunctivitis 
follows,  and  for  this  reason  alone  the  practice  is  not  to  be 
recommended  in  human  medicine. 

The  tuberculin  may  be  given  hypodermically  in  strong- 
doses,  and  this  method  again  is  not  free  from  danger. 

The  cuti-reaction  of  Von  Pirquet  is  the  most  reliable  and 
the  most  widely  used  method.  The  tuberculin  employed  is 
Koch's  "old"  tuberculin  in  25  per  cent,  strength.  A  small 
skin  area  on  both  arms  of  the  patient  is  cleansed  with  ether 
and  scarified,  as  if  for  ordinary  "vaccination,"  by  means  of 
a  sterile  surgical  needle.  The  drawing  of  blood  should  be 
avoided.  On  one  area  the  tuberculin  is  smeared,  on  the  other 
normal  saline  as  a  control.  The  lesions  are  covered  with  dry 
sterile  gauze  and  examined  after  24  hours  and  again  after 
48  hours.  There  is  no  reaction  in  normal  people,  while 
tuberculous  patients  develop  a  definite  red,  raised  papule,  with 
the  occasional  addition  of  small  vesicles. 

Von  Pirquet's  reaction  is  only  reliable  within  certain  definite 
limits.  Patients  dying  of  tuberculosis  may  not  react  at  all  and 
patients  with  healed  tuberculous  lesions  may  react  strongly. 
Owing  to  the  large  number  of  adults  with  quiescent  tuberculous 
foci  a  positive  reaction  in  an  adult  is  little  evidence  of  active 
tuberculosis.  A  negative  reaction  in  an  adult  is  evidence 
against  tuberculosis.  In  children  the  reaction,  whether 
negative  or  positive,  is  of  considerable  value. 

Other  diagnostic  reactions  are  occasionally  performed  on 
similar  lines.  A  cutaneous  and  an  ophthalmic  reaction  has 
been  made  use  of  as  a  test  for  typhoid  fever,  and  the  subcu- 
taneous injection  of  mallein  is  largely  practised  in  veterinary 
pathology  as  a  means  of  detecting  glandered  horses. 


174  CLINICAL  PATHOLOGY. 


Anti-sera. 


Anti-sera  consist  of  the  blood  sera  of  animals — in  the 
majority  of  cases  horses — which  have  been  highly  immunised 
against  bacteria  or  their  toxins.  The  injection  of  such  sera 
into  human  beings  is  the  method  of  artificially  producing  a 
passive  immunity.  The  immunity  thus  conferred  is  very 
different  from  that  aimed  at  in  vaccine  therapy,  since  the  anti- 
bodies are,  in  the  case  of  sera,  manufactured  by  the  animal  and 
injected  into  the  human  body.  The  resulting  immunity  is  in 
consequence  very  rapidly  produced  and  of  short  duration. 

The  antitoxic  sera  are  in  most  instances  standardised  on  the 
basis  of  the  amount  of  toxin  which  they  can  neutralise.  In 
the  case  of  diphtheria  antitoxin  the  smallest  amount  of  diph- 
theria toxin  capable  of  killing  a  guinea-pig  of  250  grammes 
weight  within  4  days  is  first  determined.  This  is  known 
as  the  minimum  lethal  dose.  The  amount  of  antitoxic  serum 
which  will  neutralise  100  times  the  minimum  lethal  dose  is 
found  by  mixing  serum  and  anti-serum  and  then  injecting 
the  mixture  into  a  guinea-pig  without  causing  death.  This 
amount  is  reckoned  as  1  immunity  unit  of  antitoxin. 

The  anti-sera  are  obtained  by  the  repeated  inoculation  of 
horses  with  bacteria  or  their  toxins  until  a  high  degree  of 
immunity  is  attained  in  the  horse's  serum.  The  animal  is 
then  bled  from  the  jugular  vein  into  a  sterile  receptacle.  The 
clear  serum  which  results  after  standing  or  centrifuging  is 
pipetted  off  and  stored  in  sterile  glass  capsules  in  measured 
doses.  The  anti-sera  are  given  to  human  beings  by  subcu- 
taneous injections  into  the  loose  tissues  of  the  axilla  or 
abdominal  wall.  The  dose  may  be  repeated  in  24  or  48  hours, 
but  it  is  rarely  advisable  to  give  more  than  3  doses. 

In  favourable  cases  the  temperature  drops  to  normal  within  a 
few  hours  and  the  local  and  general  condition  rapidly  improves. 

The  injection  of  serum  is  not  infrequently  followed  by  the 
appearance  of  a  diffuse  and  irritating  rash,  usually  of  the  nature 
of  urticaria  and  less  frequently  by  painful  effusions  into  the 
joints. 

The  severe  anaphylactic  phenomena  consisting  of  rapid 
collapse  and  death,  which  are  readily  induced  in  animals  by 
repeating  the  injection  of  the  serum  after  an  interval  of  2  to 
3  weeks,   are  very   rarely  observed  in  man.      The    risk   of 


VACCINES— ANTI-SEEA.  175 

giving  repeated  doses  at  small  intervals  is  almost  nil,  but  the 
procedure  is  more  liable  to  be  followed  by  rashes  and  joint 
affections  than  if  a  single  immunising  dose  is  given. 

The  preparation  of  anti-sera  is  beyond  the  scope  of  ordinary 
clinical  pathology,  since  the  use  of  large  animals  is  involved, 
and  reliable  sera  can  be  obtained  from  some  of  the  leading 
chemical  firms. 

Only  a  brief  indication  of  the  mode  of  preparation,  and  of 
the  dosage  and  use,  of  the  various  sera  can  be  given  here. 

Anti- diphtheritic  serum. — The  horse  is  injected  with  the 
toxin  of  the  diphtheria  bacillus  obtained  from  the  nitrate  of  a 
3  to  6  weeks  old  culture  of  the  organisms  in  broth.  The 
injections  are  continued  during  4  to  6  months,  and  then  stopped 
when  a  serum  of  high  potency  is  obtained.  The  dose  of  serum 
given  varies  from  2  to  10  thousand  units.  The  longer  the 
lapse  of  time  from  the  onset  of  infection  the  higher  is  the  dose 
given.  There  can  be  no  doubt  as  to  the  value  of  this  serum, 
which  should  be  given  in  all  cases  of  diphtheria. 

Anti- tetanic  serum  is  obtained  by  the  injection  of  a 
horse  with  tetanus  toxin  in  increasing  doses.  With  most  pre- 
parations a  minimum  of  25  c.c.  of  the  serum  should  be  given  as 
the  initial  dose.  The  therapeutic  effect  of  this  serum  in  man 
is  most  disappointing,  owing  to  the  fact  that  the  combination 
between  toxin  and  antitoxin  is  unable  to  take  place  in  the 
body  if  the  toxin  has  previously  combined  with  the  nerve 
tissues.  Consequently  when  symptoms  have  developed  it  is 
in  the  majority  of  cases  too  late  to  give  the  antitoxin  as  a 
curative.  It  is  advisable,  however,  to  give  the  serum  in  all 
cases  when  practicable  with  the  view  of  fixing  any  further 
toxin  that  may  be  elaborated  from  the  local  lesion,  while  the 
toxin  already  combined  in  the  nervous  system  is  combated  on 
ordinary  medical  principles. 

Anti-streptococcal  serum. — The  horse  is  immunised  by 
a  series  of  injections  of  a  single  strain  or  a  combination  of 
varieties  of  streptococci  of  increasing  virulence.  The  value  of 
this  serum  is  somewhat  problematical,  but  it  may  be  used  in 
cases  of  undoubted  streptococcal  infection,  and  may  be  given 
at  once  in  doses  of  20  c.c.  or  more  and  be  followed  by  injections 
of  the  appropriate  vaccine.  It  is  of  use  in  puerperal  infections, 
in  acute  inflammations  due  to  the  streptococcus,  and  possibly  in 
erysipelas. 


176  CLINICAL   PATHOLOGY. 

Anti-pneumococcus  serum  is  prepared  in  a  similar  way 
to  the  foregoing,  and  has  been  used  in  the  treatment  of  pneu- 
monia. The  induction  in  the  horse  of  a  high  immunity  to 
the  pneumococcus  appears  to  be  a  matter  of  considerable 
uncertainty,  and  many  of  the  sera  used  have  been  practically 
functionless. 

Anti-meningococcus  serum  may  be  given  in  10  c.c.  doses 
subcutaneously.  Flexner's  serum  is  given  intraspinally  by 
lumbar  puncture  in  80  c.c.  doses.  Good  results  have  been 
recorded  from  Flexner's  serum  in  the  American  epidemics,  but 
in  the  sporadic  cases  in  London  the  effect  has  not  been  so 
striking. 

Anti-colon  bacillus  serum  is  obtained  by  the  immunisa- 
tion of  horses  to  mixed  strains  of  colon  bacilli.  In  acute  coli 
infections  of  the  kidney  the  effect  of  the  serum  is  sometimes 
striking,  but  it  must  be  remembered  that  the  clinical  fluctua- 
tions in  this  condition  apart  from  serum  treatment  are  often 
remarkable.  The  serum  should  be  given  in  20  c.c.  doses  on  3 
consecutive  days. 

Anti-plague  serum. — The  horse  is  immunised  with  saline 
suspensions  of  the  bacilli.  The  first  doses  are  sterilised  by 
heat.  In  the  later  doses  living  bacilli  are  given.  The  serum 
is  usually  given  in  20  c.c.  doses. 

In  addition  to  the  methods  indicated  above  anti-sera  have 
been  given  by  the  mouth,  per  rectum  and  by  local  applications. 
Striking  results  have  also  been  recorded  from  similar  uses  of 
normal  horse  serum.  It  cannot  be  said  that,  with  the 
exception  of  anti-diphtheritic  serum,  the  results  obtained  with 
any  of  these  sera  have  been  particularly  encouraging.  At  the 
same  time,  in  a  number  of  individual  cases  improvement  seems 
to  definitely  follow  the  injection,  while  in  many  fatal  cases  the 
sera  have  been  given  as  a  last  resort  in  the  most  desperate 
conditions. 


CHAPTER  XIII. 

pebparation  of  culture  media staining  reagents. 

The  Preparation  of  Culture  Media. 

The  main  constituents  of  the  commoner  culture  media  and 
the  changes  which  take  place  in  the  media  as  the  result  of 
bacteriological  growth  have  been  already  referred  to.  The 
present  chapter  deals  with  the  practical  modes  of  preparation 
of  the  media. 

Sterilisation. — It  is  essential  that  all  the  media  and  the 


Fig.  12. — Hot-air  Steriliser. 

receptacles  which  contain  them  should  be  perfectly  free  from 
all  living  organisms  before  they  are  used  for  inoculation. 
The  following  apparatus  is  required  : — 

A  thermometer  graduated  to  200°  C. 
A  hot-air  steriliser. 
A  steam  steriliser. 
An  autoclave. 
An  inspissator. 
The  hot-air~steriliser  consists  of  a  double-walled  chamber 
p.  12 


178 


CLINICAL  PATHOLOGY. 


of  copper  or  iron  heated  below  by  a  gas  flame  and  fitted  with 
a  thermometer  passing  into  the  inner  chamber.  It  is  used 
for  the  sterilisation  of  glass  flasks,  test  tubes,  Petri  dishes,  etc., 
which  can  thus  be  rendered  both  sterile  and  dry.  A  tempera- 
ture of  170°  C.  for  1  hour  is  sufficient  to  destroy  any  organisms 
that  may  be  present. 

The  steam  steriliser  consists  of  a  metal  cylinder  on  legs 
enclosed  in  felt  or  asbestos  and  provided  with  a  perforated 
tray  fixed  about  8  inches  from  the  bottom.  Water  to  the 
depth  of  3  inches  is  placed  in  the  bottom. 
The  space  above  the  tray  should  be  tall 
enough  to  accommodate  a  litre  flask  fitted 
with  a  funnel.  An  ordinary  potato 
steamer  can  be  adapted  to  the  purpose. 
Media  placed  in  the  steamer  are  sur- 
rounded with  steam  at  100°  C.  The 
majority  of  media,  after  being  exposed 
to  contamination,  are  steamed  for  30 
minutes  on  3  consecutive  days. 

The  autoclave  is  used  for  rapid  and 
effective  sterilisation  by  means  of  steam 
at  high  pressure.  It  is  not  absolutely 
essential,  and  requires  careful  supervision 
when  in  use.  The  autoclave  consists  of 
a  gun-metal  cylinder  provided  with  a 
movable  or  hinged  lid,  fitted  on  with 
screws  and  nuts,  and  with  a  pressure 
gauge  and  safety  valve.  The  water 
is  usually  boiled  at  a  temperature  of 
120°  C,  which  requires  a  pressure  of 
30  lbs.  to  the  square  inch,  and  30  minutes  at  this  temperature 
is  usually  sufficient.  The  chamber  must  be  filled  with  steam 
before  beginning  to  raise  the  pressure.  The  autoclave  must 
be  allowed  to  cool  to  100°  C.  before  opening  it  or  blowing  off 
steam.  All  the  above  sterilisers  should  be  allowed  to  cool  down 
considerably  before  opening,  otherwise  the  glass  receptacles 
are  liable  to  crack. 

The  inspissator  is  required  mainly  for  the  preparation  of 
blood  serum  media.  It  consists  of  a  shallow  sloped  chamber 
provided  with  a  water  jacket  and  a  thermometer.  The  serum 
tubes  are  kept   at  75°  C.  in   the   chamber    until   the   serum 


Fig.  13.— Steam 
Steriliser. 


PEEPAEATION   OF   CULTURE   MEDIA.  179 

solidifies.  The  top  of  the  apparatus  is  of  glass  covered  with 
thick  felt,  which  can  be  lifted  for  inspection  of  the  tubes  in 
the  interior  of  the  chamber. 

All  the  receptacles,  such  as  flasks  and  test  tubes,  used  in  the 
preparation  of  media  should  be  perfectly  clean  and  free  from 


Fig.  14.— Autoclave. 

acid  or  alkali.  They  should  be  sterilised  preferably  before 
being  filled,  and  always  after  exposure  to  contamination.  The 
mouths  of  flasks  and  test  tubes  are  corked  with  plugs  of  non- 
absorbent  wool.  The  plugs  should  fill  the  mouths  of  the  tubes 
and  should  project  beyond  them,  but  should  not  be  too  tight 
nor  too  bulky.     Ordinary  cleanliness  should  be  observed  in  all 

12—2 


180 


CLINICAL   PATHOLOGY. 


operations  connected  with  media-making  in  order  to  avoid 
the  needless  introduction  of  spore-bearing  organisms.  The 
methods  of  making  the  following  media  are  given  in  brief,  and 
the  smaller  details  are  left  to  the  management  of  the  individual 
worker. 


Fig.  15. — Inspissator. 

Stock  nutrient  broth. — This  medium  is  widely  used  and 
forms  the  basis  of  many  other  media. 
Composition : — 


.     55  grammes. 
•     55 
.     55 
5,500  c.c. 


"  Lemco  "..... 
Sodium  chloride 

Peptone      ..... 
Tap  water  ..... 
To  make : — 
Boil  the  water  in  a  saucepan. 
Add  NaCl,  peptone  and  Lemco. 
Boil  for  45  minutes  and  pour  into  flasks. 
Standardise  (see  below). 
Steam  flasks  for  1  hour. 

Stand  in  cool  place  till  next  day.     Filter  when  cold. 
Either    place    in   flasks   for    stock,   and    autoclave    for 

30  minutes, 
Or,  as  required,  tube. 
To  tube,  fill  the  number  of  test  tubes  wanted  to  about  one- 
third  of  their  capacity  and  cork  with  wool  plugs. 


PREPARATION   OF   CULTURE   MEDIA.  181 

Autoclave  30  minutes  (or  steam  for  30  minutes  on  3  con- 
secutive days). 

The  standardisation  is  carried  out  as  follows  : — 
Take  10  c.c.  of  the  stock  broth. 
Add  a  few  drops  of  phenol-phthalein. 
Carefully  run  in  deci-normal  caustic  soda  solution  until  a 

faint  permanent  pink  colour  is  produced. 
Read   off  the   amount   of    soda  used  and   calculate   the 
amount  of  normal  soda  required  to  neutralise  1  litre 
of  the  broth  to  phenol-phthalein. 
The  amount  required  is  commonly  about  15  c.c. 
The  broth  is  as  a  rule  not  exactly  neutralised,  but  is  made 
up  to  a  definite  known  acidity.     The  acidity  usually  chosen  is 
that  known  as  +  5  on  Eyre's  scale,  and  is  a  favourable  one  for 
the  growth  of  the  majority  of  organisms,     By  an  acidity  of 
+  5  is  meant  that  the  normal  soda  added  per  litre  is  5  c.c.  less 
than  that  required  to  exactly  neutralise  to  the  phenol-phthalein 
end  point.     For  example,  if  10  c.c.   of   the   broth  required 

N 
1*5  c.c.  of  ryr  soda  to  neutralise,  1  litre  would  require  150  c.c. 

of  r^  soda,  or  15  c.c.  of  N  soda.     In  such  a  case  10  c.c.  of 

N  soda  per  litre  are  added,  leaving  unneutralised  an  acidity  of 
—  5  c.c.  N  soda,  or  +  5  on  Eyre's  scale.  If  an  acidity  of  —  10 
is  required  (an  acidity  recommended  for  the  growth  of  some 
streptococci),  25  c.c.  of  N  soda  are  added  per  litre.  A  broth 
solution  with  an  acidity  of  +  5  is  alkaline  to  litmus. 

Glycerine  broth  is  made  by  the  addition  of  6  per  cent,  of 
glycerine  to  the  stock  nutrient  broth. 

Glucose  broth  is  made  by  the  addition  of  2  per  cent,  of 
glucose  to  the  stock  nutrient  broth. 

Neutral  red  broth  is  made  by  adding  2  c.c.  of  a  2  per 
cent,  aqueous  solution  of  neutral  red  to  100  c.c.  of  nutrient 
broth. 

Litmus  carbohydrate  broths  are  made  by  adding 
1  gramme  of  the  carbohydrate  to  90  c.c.  of  nutrient  broth, 
which  has  been  neutralised  to  phenol-phthalein,  and  10  c.c. 
of  litmus  solution.  The  carbohydrates  most  commonly 
required  are  dextrose,  lactose,  mannite,  ramnose,  and  salicin. 
Others  which  may  be  employed  are  arabinose,  saccharoso, 
inulin,  coniferin,  sorbit,  etc. 


182  CLINICAL  PATHOLOGY. 

The  litmus  solution  required  to  give  the  necessary  colour 
may  be  bought  ready  made  or  prepared  as  follows  : — 
Materials  required : — 

Litmus  powder 20  grammes. 

90  per  cent,  alcohol    ....  200  c.c. 

Distilled  water 200    „ 

To  make  : — 

Boil  the  litmus  with  80  c.c.  of  the  alcohol  for  1  hour  on  a 

water  bath. 
Pour  off  the  clear  liquid. 
Eepeat  with  60  c.c.  of  the  alcohol. 
Eepeat  with  the  remainder  of  the  alcohol. 
Digest  the  washed  litmus  in  the  distilled  water. 
Filter. 
When  tubing  the  carbohydrate   media  it  is  convenient  to 
place  in  each  test  tube  a  small  inverted  tube  filled  with  the 
medium  in  order  to  collect  the  gas  which  may  be  evolved  as 
the  result  of  bacterial  growth. 

Agar-agar. — Agar  medium  consists  of  a  solution  of  agar- 
agar  in  stock  nutrient  broth.     It  is  best  prepared  from  the 
powdered  substance,  and  has  the  following  composition  : — 
Agar  .......     30  grammes. 

Nutrient  broth,  ....         1,000  c.c. 

To  make : — 

Dissolve  the  agar  in  the  broth  by  heating  in  the  auto- 
clave for  15  minutes  at  110°  C. 
Standardise  while  hot  (+  5  on  Eyre's  scale). 
Cool  to  60°  C. 
Add  the  beaten  white  of  1   egg.     (This  clears  the  mixture, 
but  is  usually  unnecessary  if  agar  powder  is  used  instead  of 
the  fibre.) 

Autoclave  for  20  minutes  at  115°  C. 
Filter  through  a  Chardin  filter  paper  while  hot. 
Tube  if  required. 

Steam  for  1  hour  on  3  consecutive  days. 
The  tubes  should  be  prepared  in  two  forms — for  "  stab  " 
cultures,  in  which  case  the  tubes  are  filled  to  about  two-thirds 
their  capacity  and  allowed  to  cool  in  the  vertical  position,  and 
for  "  slope"  cultures,  in  which  case  they  are  filled  about  one- 
sixth  full  and  allowed  to  cool  in  a  slanting  position. 
MacConkey's  neutral  red  agar. — This  medium  is  made 


PREPARATION  OF  CULTURE  MEDIA.    183 

by  adding  the  appropriate  substances  to  the  stock  agar.     It 
has  the  following  composition  : — 

Agar 30  grammes. 

Lactose         ......     10         „ 

Sodium  taurocholate     .  .         .5         „ 

2  per  cent,  aqueous  neutral  red  solution     20  c.c. 
Nutrient  broth  to  1  litre. 
Nasgar. — This  medium  consists  of  nutrose,  ascitic  fluid 
(or  blood  serum),  and  agar. 
It  is  made  as  follows  : — 

Take     Ascitic  fluid        .         .         .         .15     c.c. 

Nutrose     .....       1     gramme. 

Agar  powder      ....       0*5         ,, 

Distilled  water  .         .         .         .     35     c.c. 

Bring  gradually  to  the  boil  and  filter. 
Add  1  part  of  this  mixture  to  2  parts  of  agar. 
Mix  at  60°  C. 

Standardise  (+  5  on  Eyre's  scale). 
Autoclave  20  minutes  at  115°  C. 
Filter. 

Tube  (in  slope  form)  as  required  and  steam. 
Oleic  acid  agar. — This  medium  has  the  following  com- 
position : — 

Glycerine 2     per  cent. 

Oleic  acid  ......     0"1         „ 

Added  to  neutral  agar  medium. 
Gelatin. — Gelatin  media  consist  of  broth  with  sufficient 
gelatin  dissolved  in  it  to  produce  a  solid  medium  at  tenrpera- 
tures  from  18°  to  22°  C.  The  necessary  proportion  of  gelatin 
to  broth  is  25  per  cent,  of  the  former  during  the  summer 
and  20  per  cent,  during  the  winter.  It  is  important  to 
obtain  the  best  gelatin,  otherwise  a  soft  medium  will  result. 
Coignet's  gold  label  gelatin  is  very  satisfactory. 
To  make  : — 

Add  the  gelatin  to  warm  nutrient  broth. 
Dissolve  while  warm. 

Standardise  while  warm  (-f-  5  on  Eyre's  scale). 
Add  beaten  white  of  1  egg. 
Filter. 
Tube  as  required.     (Put  up  both  slope  and  stab  media.) 
Litmus    milk. — The    milk    must    be    quite    fresh,   and 


i84  CLINICAL  PATHOLOGY. 

preferably  the  majority  of  the  cream  should  have  been 
removed. 

Steam  for  30  minutes. 

Filter. 

Add  10  to  15  per  cent,  of  the  stock  litmus  solution. 

Add  the  minimum  quantity  of  normal  soda  necessary  to  give 
a  definite  blue  colour  to  the  litmus  (i.e.,  0'5  to  l'O  per  cent.). 

Tube. 

Steam  the  tubes  for  1  hour  on  3  consecutive  days. 

Potato  medium. — This  medium  is  much  less  commonly 
used  than  formerly.  Special  test  tubes  are  required,  which 
resemble  Buchner's  anaerobic  tubes  in  miniature.  A  few 
drops  of  glycerine  are  placed  in  the  bottoms  of  the  tubes,  and 
sticks  of  potato  well  washed  are  cut  and  passed  into  the  tubes, 
which  are  plugged  with  wool  and  steamed  for  1  hour  on  3 
consecutive  days.  Unless  used  at  once,  it  is  preferable  to 
cover  the  wool  plugs  with  rubber  caps. 

Dorset's  egg  medium. — This  medium  is  used  for  the 
growth  of  the  tubercle  bacillus,  and  is  prepared  as  follows  : — 

Four  eggs  are  well  beaten  up,  25  c.c.  of  water  are  added, 
and  the  whole  is  thoroughly  mixed.  The  mixture  is  passed 
through  muslin  and  tubed.  The  tubes  in  the  form  of  slope 
cultures  are  placed  in  the  inspissator  and  steamed  for  4  hours 
at  70°  C.     They  are  then  sterilised  in  the  usual  way. 

Blood  serum  media. — Blood,  preferably  that  of  a  bullock, 
is  obtained  at  the  slaughter-house,  and  is  taken  from  the 
wound,  after  a  little  blood  has  been  allowed  to  flow,  direct  into 
a  large  sterile  glass  cylinder.  The  cylinder  is  placed  on  ice 
until  the  serum  has  fully  separated,  that  is,  as  a  rule,  until 
the  next  day. 

With  a  sterile  glass  pipette  the  serum  is  sucked  up  from 
the  cylinder  and  blown  out  into  sterile  test  tubes.  The  serum 
is  often  tinged  red  from  haemolysis  of  the  cells,  but  this  is  of 
no  importance  and  the  colour  goes  in  the  subsequent  processes. 
The  tubes  are  then  placed  in  a  sloped  position  in  the  inspissa- 
tor and  kept  at  a  temperature  of  75°  C.  for  about  4  hours,  or 
until  they  have  become  firmly  set.  The  heating  is  repeated 
for  1  hour  only  on  each  of  the  2  consecutive  days.  The 
media  thus  prepared  are  almost  invariably  sterile,  but  it  is 
customary  to  incubate  them  for  24  hours  in  order  to  prove 
their  sterility. 


PEEPAEATION   OF   CULTUEE   MEDIA.  185 

Instead  of  ox  blood  that  of  the  sheep  can  be  used,  or  human 
blood  obtained  at  venesection. 

Hydrocele  fluid  or  ascitic  fluid  can  be  made  use  of  in  the 
same  manner. 

Loffler's  blood  serum  has  the  following  composition : — 

Four  parts  of  serum  to  1  part  of  nutrient  broth  with  1  per 
cent,  of  glucose  added.  The  mixture  is  made  before  the  tubes 
are  filled,  and  a  rather  higher  temperature  may  be  required 
to  obtain  a  satisfactory  solidification  of  the  medium. 

Ox-bile  medium. — This  medium  is  used  for  the  growth  of 
the  typhoid  bacillus,  and  is  particularly  useful  if  blood  in  the 
early  days  of  typhoid  fever  has  to  be  obtained  from  the  finger. 
The  typhoid  bacilli  grow  readily  in  this  medium  and  the 
common  skin  cocci  do  not. 

The  gall-bladder  of  an  ox  is  tied  off  and  removed  at  the 
slaughter-house ;  100  c.c.  of  bile  are  mixed  with  10  grammes  of 
peptone  and  2  grammes  of  sodium  chloride. 

The  mixture  is  autoclaved  for  15  minutes  at  120°  C,  filtered 
and  tubed. 

The  sterilisation  of  inoculated  media. — It  is  necessary  to 
destroy  the  organisms  which  have  grown  in  the  discarded  culture 
media  and  to  render  the  test  tubes  and  plates  fit  for  further  use. 

Test  tubes. — Place  culture  tubes  without  removing  the 
wool  plugs  into  a  large  saucepan  with  a  well-fitting  lid. 

Bring  to  boiling  point  and  boil  for  3  hours. 

Eepeat  boiling  on  the  following  day. 

When  cold  remove  plugs.  Empty  contents  down  sink  and 
put  tubes  in  a  basin  with  hot  water  running  over  them. 

Wash  with  test-tube  brush,  using  25  per  cent.  HC1. 

Wash  with  cold  water  several  times  to  remove  acid. 

Place  upside  down  in  crates  in  a  warm  oven  to  dry  and 
leave  for  several  hours. 

Petri  dishes. — Sterilise  by  boiling  in  the  same  way  as  the 
tubes. 

Place  in  a  solution  of  hot  strong  Hudson's  soap  water  for 
half  an  hour,  after  separating  the  plates. 

Wash  thoroughly  in  the  water. 

Wash  with  clear  cold  water. 

Wipe  dry. 

Sterilise  in  hot  air  steriliser  for  60  minutes  at  170°  C. 

(Washing  soda  must  not  be  used.) 


186  CLINICAL   PATHOLOGY. 

To  clean  used  microscopic  slides. — Soak  the  slides  in 
cold  water  to  remove  the  lysol. 

Boil  for  1  hour  in  Hudson's  soap  water. 
Take  out  and  remove  cover-glasses. 
Boil  again  for  15  minutes. 
Wash  in  hot  water. 

The  Preparation  op  Staining  Reagents. 

The  number  of  staining  reagents  required  for  the  ordinary 
routine  methods  of  clinical  pathology  is  very  small.  Only  the 
f  orrnulse  of  the  essential  stains  are  given  here,  and  it  is  remark- 
able how  rarely  one  requires  any  stain  which  is  not  to  be 
found  in  the  scanty  array  of  drop  bottles  disposable  on  one 
small  shelf. 

The  stains  given  here  include  those  required  for  ordinary 
histological  purposes  as  well  as  for  bacteriological  work.  The 
special  blood  stains  are  described  in  the  section  on  the  blood, 
and  a  few  other  special  stains  are  given  in  their  appropriate 
places  in  the  text. 

Carbol  thionin. — This  is  the  most  useful  general  stain 
in  routine  bacteriological  work.  It  has  the  following 
formula : — 

Saturated  solution  of  thionin  in  50  per 

cent,  alcohol      .....     10  cc. 
Carbolic  acid  (crystal)  ....       1  gramme. 

Distilled  water 90  cc. 

The  saturated  alcoholic  solution  of  thionin  requires  about 
5  per  cent,  of  thionin,  and  should  be  kept  in  the  incubator 
at  37°  C.  for  one  week.  It  is  then  filtered,  kept  in  stock, 
and  made  up  as  required  with  the  carbolic  acid  and 
water. 

Methylene  blue. — A  saturated  watery  solution  has  the 
following  composition : — 

Methylene  blue  (Griibler)    .         .  about  20  grammes. 

Distilled  water 400  cc. 

The  stain  is  added  to  the  distilled  water  in  a  bottle  with  a 

well-fitted  stopper,  and  the  bottle  is  kept  in  a  hot  oven  (or  in 

the   incubator  at  37°  C);  for  several  days.     If  all  the  stain 

is  dissolved  more  is  added  until  the  mixture  is  saturated. 

A  saturated  alcoholic  solution  is  made  in  the  same  way  by 


PEEPAEATION  OP  CULTURE  MEDIA.    187 

saturating  about  5  grammes  of  methylene  blue  in  100  c.c.  of 
absolute  alcohol. 

The  saturated  watery  and  alcoholic  solutions  are  kept  as 
stock,  and  a  1  per  cent,  dilution  of  the   watery  solution  is 
used  for  ordinary  staining  purposes.    The  alcoholic  solution  is 
required  for  the  next  stain. 
Lb'ffler's  methylene  blue. — Formula  : — 
Saturated   solution   of  methylene   blue  in 

alcohol    .......     30  c.c. 

Caustic  potash  1  per  cent 1    „ 

Distilled  water    ......  100    „ 

Carbol  fuchsin. — Formula  :  — 

Basic  fuchsin  (Griibler)       ...       1  gramme. 
Absolute  alcohol  .         .         .         .10  c.c. 

Carbolic  acid  1  in  20  .         .         .         .  100  c.c. 

The    stain  is   shaken  with   the   alcohol   and   the    carbolic 
added  to  it.     This  solution  is  kept  as  stock  and  is  filtered  into 
drop  bottles  as  required. 

Gentian  violet  (alcoholic). — Formula  : — 

Gentian  violet         .         .         .       about  10  grammes. 
Absolute  alcohol     ....       300  cc. 

The  mixture  is  kept  on  an  oven  or  in  the  incubator  for  one 
week,  and  if  not  saturated  at  the  end  of  this  time  more  gentian 
violet  is  added. 

This  solution  is  kept  as  stock  and  filtered  before  use. 
Gram's  iodine. — Formula  : — 

Iodine        ......       1  gramme. 

Potassium  iodide        ....       2  grammes. 

Distilled  water 300  c.c. 

A  clear  solution  results,  which  does  not  require  filtering. 
Giemsa's  stain. — Formula  : — 

Azur  ii.  eosin  .....         3  parts. 

Azur  ii.  .         .         .         .         .         .         .      0*8      ,, 

Glycerin  (Merck  pure)    ....    250      ,, 

Methyl  alcohol  (Kahlbaum)    .         .         .    250      ,, 
Safranin. — This  is  a  useful  counter-stain  to  Gram's  method 
for  organisms  in  sections. 
Formula : — 

Safranin  .....       0'5  gramme. 

Distilled  water         ....      100  c.c. 

The  stain  is  filtered  on  the  section  when  used. 


188  CLINICAL  PATHOLOGY. 

Hsemalum. — This  is  the  most  universal  nuclear  stain  for 
tissues.  It  is  also  convenient  for  the  staining  of  leucocyte 
drops  by  Strong's  method. 

Formula  : — 

1.  Pure  hffiinatein  ....         2\5  grammes. 
Alcohol  95  per  cent.    ...  95  c.c. 

Dissolve 

2.  Ammonium  alum        .         .         .       125  grammes. 
Distilled  water    ....    2,000  c.c. 

Dissolve. 

Add  1  to  2. 

Add  to  the  mixture 

Glacial  acetic  acid      .....     325  c.c. 

Shake  well  and  allow  to  ripen  on  a  shelf  in  the  light,  if 
possible  for  from  1  to  3  months. 

Filter  before  use. 

Eosin. — This  is  an  excellent  tissue  stain  for  sections. 

Alcoholic  solution.  A  saturated  solution  is  made  in  methy- 
lated spirit.  About  2  grammes  are  required  for  100  c.c.  of 
spirit.  Five  per  cent,  of  this  solution  in  distilled  water  or 
spirit  is  used  for  staining  purposes. 

Watery  solution.  The  saturated  solution  is  made  in 
distilled  water.  About  10  grammes  are  required  for  100  c.c. 
of  water. 

Two  per  cent,  of  this  solution  in  distilled  water  is  used. 

Van  Gieson's  stain. — This  stain  is  a  very  widely  used 
differential  stain  for  histological  purposes.  It  has  the  follow- 
ing composition : — 

Saturated  watery  solution  of  picric  acid        .     50  c.c. 
Watery  solution  of  acid  fuchsin  1  per  cent.  .     15    ,, 
Distilled  water      .         .         .         .         .         .     50    ,, 

Scharlach  R. — This  stain  is  the  most  satisfactory  for  the 
demonstration  of  fat  in  tissues  or  in  film  preparations.  Fat 
globules  are  stained  red  by  it,  and  all  other  tissues  are  left 
unstained.     It  is  prepared  as  follows  : — 

Put  100  c.c.  of  75  per  cent,  absolute  alcohol  in  distilled 
water  into  a  bottle.  Add  Scharlach  E.  Shake  vigorously  and 
allow  to  stand  for  several  days  in  the  cold.  When  the  fluid  is 
saturated  filter  it. 


SECTION   III. 

PUNCTUKE    FLUIDS. 

CHAPTER  XIV. 
General  Procedure— Pleural  Fluids — Pericardial  Fluids. 

CHAPTER  XV. 

Peritoneal  Fluids — Cerebro-spinal  Fluids — Synovial  Fluids  —Cysts,  etc. 


CHAPTER  XIV. 

GENERAL    PROCEDURE PLEURAL    FLUIDS — PERICARDIAL 

FLUIDS. 

By  puncture  fluids  are  meant  such  body  fluids,  whether 
exudates,  transudates,  or  the  contents  of  cysts,  as  are 
commonly  removed  for  examination  by  means  of  a  needle  and 
syringe. 

The  fluids  most  frequently  taken  for  investigation  are  those 
derived  from  the  pleural  and  peritoneal  cavities  and  from  the 
cerebro- spinal  canal. 

No  attempt  is  made  here  to  give  a  detailed  account  of  the 
complete  examination  of  these  fluids.  We  are  concerned  only 
with  such  investigations  as  reveal  the  nature  of  the  patho- 
logical processes  producing  the  effusions  and  such  as  are  of 
practical  value  in  clinical  medicine.  The  complete  chemical 
analysis  of  any  exudate  has  a  valuable  scientific  interest,  but 
requires  very  many  hours  of  laborious  work,  which  is  at  the 
present  of  the  nature  of  experimental  research  rather  than 
of  practical  clinical  value.  With  the  majority  of  fluids  we 
depend  mainly  upon  determining  the  nature  of  the  cells  and 
bacteria  present  in  them,  and  can  by  these  means  in  almost 
every  case  give  to  the  clinician  an  accurate  diagnosis  of  the 
nature  of  the  disease.  Purely  chemical  tests  are  in  this 
connection  of  little  value  to  us. 

General  Procedure. 

The  procedure  varies  somewhat  according  as  the  fluid  to  be 
examined  is  obviously  purulent  or  not.  In  the  case  of  puru- 
lent exudates  the  examination  is  conducted  in  a  precisely 
similar  manner  to  that  employed  for  the  investigation  of  pus 
from  any  other  part  of  the  body. 

In  the  case  of  fluids  not  obviously  purulent  the  procedure 
is  as  follows  : — 

(1)  Inspection. — The  naked-eye  appearance  of  these  fluids 
is  often  of  considerable  help.    It  should  be  noted  whether  they 


GENERAL   PROCEDURE— FLUIDS.  191 

are  clear,  turbid,  or  flocculent ;  if  they  contain  blood,  and,  if  so, 
whether  the  blood  is  in  great  or  small  amount,  and  intimately 
mixed  or  not ;  if  a  clot  forms  on  standing,  and  the  appear- 
ance of  the  clot. 

(2)  Chemical  examination. — This  can  be  mainly  omitted 
in  the  majority  of  cases  so  far  as  clinical  diagnosis  is 
concerned.  The  only  tests  that  need  be  employed  are  for 
the  reaction,  the  specific  gravity,  the  amount  of  coagulable 
proteid,  and  the  amount  of  globulin.  The  globulin  or  proteid 
content  of  cerebro-spinal  fluid  in  particular  is  of  diagnostic 
significance.  The  other  tests  are  rarely  necessary  for 
diagnosis. 

(3)  Cytology. — By  cyto-diagnosis  is  meant  an  investiga- 
tion of  the  number  and  nature  of  the  cells  present  in  a 
fluid.  Such  investigation  should  be  made  as  soon  as  possible 
after  the  fluid  has  been  withdrawn. 

The  cells  which  may  be  present  in  these  fluids  are  mostly 
of  three  varieties,  namely,  small  lymphocytes,  polynuclear 
neutrophils,  and  endothelial  cells.  A  relative  predominance 
of  one  of  these  cells,  when  present  in  excess,  is  among  the  most 
reliable  diagnostic  signs  in  clinical  pathology. 

Small  lymphocytes  in  excess  are  diagnostic  of  the 
chronic  infective  granulomata,  of  which  syphilis  and  tuber- 
culosis are  by  far  the  most  important  examples.  The  clinical 
distinction  between  syphilis  and  tuberculosis  in  the  case  of 
the  central  nervous  system  is  in  the  great  majority  of  cases 
clear ;  syphilis  of  the  pleural  and  peritoneal  sacs  is  an 
extremely  rare  pathological  condition ;  consequently  the 
demonstration  of  an  excess  of  small  lymphocytes  in  any  of 
these  fluids  commonly  suffices  to  definitely  substantiate  a 
diagnosis. 

These  small  lymphocytes  or,  as  they  are  called  by  some, 
lymphoid  cells,  cannot  be  distinguished  on  histological  grounds 
from  the  small  lymphocyte  of  the  blood,  the  cell  which  is 
relatively  increased  in  the  circulating  blood  in  tuberculosis 
and  syphilis.  The  cell  is  also  probably  identical  with  the 
lymphoid  cells  found  in  the  tissues  in  large  numbers  in  the 
neighbourhood  of  tuberculous,  and  to  a  less  obvious  extent 
of  syphilitic,  lesions.  The  part  played  by  the  lymphoid  cells 
is  obscure,  but  it  is  evident  that  this  is  the  type  of  cell  which 
is  actively  attracted  into  the  blood,  and  from  the  blood  into 


192  CLINICAL   PATHOLOGY. 

the  exudates  and  tissues,  by  the  toxins  of  the  tubercle  bacillus 
and  the  spirocheeta  pallida. 

Polynuclear  neutrophils. — The  presence  of  these  cells  is 
definite  evidence  of  an  acute  inflammatory  process.  Acute 
inflammation  of  the  serous  sacs  or  of  the  cerebro-spinal  canal 
may  be  induced  by  bacteria  or  by  an  aseptic  irritant  acting  as 
a  foreign  body.  In  human  pathology  the  exciting  cause  is  in 
the  vast  majority  of  cases  a  pyogenic  organism.  The  demon- 
stration of  polynuclear  neutrophils  therefore  calls  for  a  further 
examination  by  microscopical  and  cultural  methods  in  order 
to  identify  the  causative  organism.  The  distinction  between 
clear,  turbid,  and  obviously  purulent  fluids  all  containing  the 
polynuclear  neutrophil  as  the  predominant  cell  is  only  one  of 
degree.  The  process  has  in  each  case  the  same  pathological 
basis.  For  example,  a  clear  or  faintly  turbid  fluid  with- 
drawn from  the  chest  after  an  attack  of  lobar  pneumonia  may 
be  shown  on  careful  examination  to  contain  both  polynuclears 
and  pneumococci,  and  thus  to  be  of  the  same  nature  as  the 
thick  pus  of  an  ordinary  empyema.  When  blood  is  present 
the  demonstration  of  an  occasional  polynuclear  neutrophil  is 
of  no  significance,  and  one  has  to  judge  in  such  cases  whether 
the  leucocytic  content  of  the  deposit  is  in  excess  of  what 
is  natural  to  the  number  of  red  cells,  remembering  that  in 
normal  blood  the  proportion  of  white  cells  to  red  is  only 
about  1  to  1,000. 

Endothelial  cells. — These  cells  are  derived  from  the 
lining  membranes  of  the  serous  cavities  and  their  presence  is 
evidence  of  a  passive  transudate.  They  are  rarely  found, 
except  in  very  small  numbers,  in  cerebro-spinal  fluid.  They 
may  be  very  numerous  in  pleural  or  peritoneal  fluids.  The 
cells  are  of  much  the  same  character  in  all  situations  in  the 
body,  and  consist  of  large,  more  or  less  rounded  cells,  the 
cytoplasm  of  which  is  basophilic  and  often  vacuolated.  The 
edges  of  the  cells  are  frequently  frayed  and  irregular.  The 
nucleus  contains  as  a  rule  two  or  three  well-marked  nucleoli. 
The  endothelial  cells  are  phagocytic  and  may  contain  red  cells, 
and,  in  the  case  of  mixed  cellular  deposits,  lymphocytes, 
polynuclear  cells,  or  bacteria.  They  often  occur  in  plaques  of 
considerable  size.  In  a  good  preparation  these  cells  are  quite 
unmistakable,  but  if  the  film  has  been  made  too  thick  the  cells 
may  be  so  shrunken  as  to  be  almost  indistinguishable  from. 


PLATE  X. 


Small  Lymphocytes.  Endothelial  Cells. 

(Tuberculous  Pleural  Exudate.)  (Peritoneal  Fluid  :  Cirrhosis  of  Liver.) 

(Leishman's  Stain.)  (Leishman's  Stain.) 


Polynuclear  Neutrophils  and  Meningococci.  Polynuclear  Neutrophils  and  Pneumococei. 

(Cerebro-spinal  Fluid.)  (Pleural  Exudate.) 

(Leishman's  Stain.)  (Carbol-thionin.) 

The  Cells  of  Puncture  Fluids. 


PLATE    X. 


GENEKAL   PROCEDURE— FLUIDS.  193 

small  lymphocytes.  In  such  cases  the  nature  of  the  cells  is 
commonly  revealed  by  examining  the  edge  of  the  film  where 
the  fluid  is  thinner  and  the  cells  have  dried  more  quickly  and 
have  consequently  shrunk  less. 

An  excess  of  endothelial  cells  is  to  be  found  in  a  variety  of 
conditions,  including  renal  disease,  general  cardiac  failure,  and 
any  local  obstruction  to  the  circulation,  as  in  malignant  disease 
or  cirrhosis  of  the  liver. 

Other  cells. — Almost  any  variety  of  cell  may  on  rare 
occasions  be  found  in  these  fluids.  The  eosinophil  cell  is 
exceptionally  predominant,  and  is  of  doubtful  significance.  It 
may  be  found  in  parasitic  effusions,  as  in  the  content  of  a 
hydatid  cyst,  or  after  the  rupture  of  a  cyst  into  the  pleural  or 
peritoneal  cavities.  It  may  form  a  considerable  percentage  of 
the  cells  present  in  the  clear  exudates  which  occasionally 
follow  lobar  pneumonia,  and  is  then  of  good  prognostic 
significance,  since  such  cases  apparently  do  not  proceed  to 
suppuration.  The  eosinophil  has  also  been  found  in  some 
numbers  in  tuberculous  exudates. 

The  large  hyaline  is  never  the  predominant  cell,  but  is  not 
infrequently  found  in  small  numbers,  particularly  in  associa- 
tion with  the  polynuclear  neutrophil. 

Malignant  cells,  whether  sarcomatous  or  carcinomatous, 
are  frequently  described  and  practically  never  identified.  The 
cells  which  are  sometimes  figured  as  sarcoma  cells  are  either 
small  lymphocytes  or  quite  indistinguishable  from  them.  The 
cells  often  described  as  carcinoma  cells  are  in  every  way 
similar  to  endothelials.  It  is  true  that  fragments  of  growth 
may  be  washed  off  into  a  serous  fluid  and  may  very  rarely 
be  identified  microscopically,  but  in  the  great  majority  of 
malignant  cases  the  predominant  cell  in  an  exudate  is  not  to 
be  distinguished  from  either  an  endothelial  cell,  or  much  less 
commonly,  a  small  lymphocyte.  In  the  routine  examination 
of  a  very  large  number  of  serous  effusions  I  have  only  once 
recognised  with  any  certainty  the  cells  of  a  malignant  growth. 
It  has  not  been  sufficiently  insisted  upon  that  a  carcinoma  or 
sarcoma  cell  floating  free  has  no  distinguishing  marks  by 
which  it  can  certainly  be  identified. 

(4)  Bacteriology. — The  method  of  bacteriological  investi- 
gation necessarily  varies  with  the  type  of  cell  present  in  the 
exudate,  and  consequently  a  portion   of   the  fluid  should  be 

p.  13 


194  CLINICAL   PATHOLOGY. 

examined  cytologically  before  an  attempt  is  made  to  discover 
the  presence  of  a  causative  organism. 

If  the  predominant  cell  is  an  endothelial  no  bacteriological 
investigation  is  necessary,  since  there  is  no  known  organism 
which  leads  to  an  over-production  of  this  type  of  cell,  at  any 
rate  in  preponderating  numbers.  The  presence  of  bacteria  in 
such  fluids  is  due  to  a  contamination  either  from  the  patient's 
skin  or  from  the  needle  and  syringe. 

When  the  predominant  cell  is  a  polynuclear  neutrophil  the 
bacteriological  investigation  must  be  conducted  on  exactly  the 
same  lines  as  that  for  acute  inflammatory  exudates  in  any  part 
of  the  body.     The  causative  organisms  themselves  may  often 
be    seen   in    the   film   preparations  made  primarily   for    the 
identification  of  the  cells,  and  cultures  are  then  made  upon  the 
appropriate  media.     If  no  organisms  are  seen  a  growth  may 
still  be  obtained  in  the  cultures,  which  should  be  made  on  media 
suitable  to  the  organism  suspected  from  the  nature  of  the  case. 
In  a  small  proportion  of  acute  inflammatory  exudates  organisms 
may  be  seen  in  the  films,  but,  owing  probably  to  the  previous 
exposure  of  the  bacteria  in  the  body  to  bacteriolytic  substances, 
the  attempt  to  grow  them  in  culture  media  fails.     In   such 
cases  a  fairly  accurate  bacteriological  diagnosis  can  usually  be 
made  from   the   nature   of    the   case  and  the  morphological 
appearance  of  the  organisms.     The  pneumococcus  in  pleural 
or   other   exudates,  and  the  meningococcus  in  cerebro-spinal 
fluids,  not  infrequently  stain  very  faintly,  and  sometimes  fail 
to  grow  upon  the  ordinary  media. 

In  another  small  percentage  of  cases  polynuclear  neutrophils 
are  the  predominant  cell,  but  no  organisms  are  seen  in  the 
film  preparations  and  none  grown  in  culture.  Some  of  these 
fluids  are  in  reality  produced  by  pyogenic  organisms  which  have 
escaped  recognition.  Others  are  not  the  product  of  organisms 
but  of  some  mechanical  irritation,  such  as  an  injury  or  the 
presence  of  new  growth,  or  an  effusion  of  blood  acting  as 
a  foreign  body.  Others,  again,  may  turn  out  to  be  tuberculous 
effusions  in  which  the  predominant  lymphoid  cell  has 
degenerated  and  the  clearly  staining  polynuclear  neutrophil, 
which  is  acting  as  a  phagocyte  of  the  cellular  debris,  appears 
to  predominate.  Primary  tuberculous  effusions  may  further 
become  secondarily  infected  by  pyogenic  organisms,  and  in 
such  cases  the  underlying  pathological  process  may  be  entirely 


GENERAL   PROCEDURE— FLUIDS.  195 

missed,  unless  a  knowledge  of  the  clinical  condition  of  the 
patient  suggests  that  the  tubercle  bacillus  also  should  be 
specially  sought  for. 

When  the  small  lymphocyte  is  the  predominant  cell  there  is 
no  object  in  putting  up  the  ordinary  culture  media  and  search- 
ing for  the  presence  of  pyogenic  organisms.  Any  bacteria 
which  may  be  seen  in  the  ordinary  film  preparations  or  grown 
on  the  stock  media  are  evidence  of  failure  in  the  aseptic 
technique.  When  an  excess  of  small  lymphocytes  with  a 
relative  predominance  of  80  per  cent,  or  more  over  the  other 
cells  is  found,  a  diagnosis  of  either  a  syphilitic  or  a  tuberculous 
infection  can  be  made  with  a  considerable  degree  of  confidence. 
Since  on  clinical  grounds  the  differential  diagnosis  between 
these  two  pathological  processes  can  in  the  great  majority  of 
cases  be  readily  made,  the  laboratory  diagnosis  of  a  lympho- 
cytic effusion  is  usually  sufficient.  Such  a  diagnosis,  however, 
is  an  indirect  one,  and  thus  liable  to  a  small  but  definite 
percentage  of  errors ;  consequently  further  investigation  is 
preferable  whenever  practicable.  In  syphilitic  cases  it  is 
useless  to  look  for  the  spirochete  in  the  serous  fluids,  but  a 
Wassermann  reaction  should  be  performed  with  the  patient's 
serum,  or  with  the  fluid,  or  with  both.  In  the  case  of  tuber- 
culous effusions  an  attempt  should  be  made  to  identify  the 
bacillus,  but  unfortunately  in  the  majority  of  these  fluids  the 
bacilli  are  extremely  scanty  and  require  special  methods  to 
display  them,  such  as  will  be  described  subsequently. 

A  small  percentage  of  lymphocytic  effusions  are  neither 
tuberculous  nor  syphilitic.  Very  occasionally  miliary  growths 
of  carcinoma  or  sarcoma  on  the  pleural  membranes,  or  less 
commonly  on  the  peritoneum,  may  act  as  chronic  irritants  and 
produce  an  exudate  rich  in  lymphoid  cells.  Such  exudates  are 
liable  to  be  mistaken,  both  on  clinical  and  pathological  grounds, 
for  tuberculous  exudates.  Similar  exudates  may  accompany 
an  aneurysm  of  the  thoracic  or  abdominal  aorta,  or  an  enlarge- 
ment of  the  mediastinal  glands  by  new  growth,  or  Hodgkin's 
disease.  All  these  rare  pathological  states  are  more  commonly 
associated  with  an  excess  of  endothelial  cells,  and  the 
percentage  of  error  in  ly mphocy  tic  effusions  is  very  small  ;  it  is 
sufficient,  however,  to  remind  one  that  no  single  physical  sign 
in  medical  diagnosis  is  pathognomonic.  An  instance  may  be 
given  of  a  pleural  effusion  rich  in  small  lymphocytes,  a  portion 

13—2 


196  CLINICAL   PATHOLOGY. 

of  which  was  injected  into  a  guinea-pig.  The  animal 
subsequently  died  of  general  tuberculosis.  The  patient  died 
shortly  afterwards,  and  no  evidence  of  tuberculous  disease 
could  anywhere  be  found.  The  probable  explanation  is  that  a 
guinea-pig  already  tuberculous  was  inoculated  with  the  sterile 
fluid.  The  incident  is  merely  quoted  as  an  example  of 
ordinary  human  fallibility  when  dealing  with  circumstantial 
evidence. 

Methods. — The  methods  to  be  adopted  in  the  examination 
of  the  various  fluids  do  not  materially  differ,  but  it  is 
convenient  for  the  sake  of  clearness  to  describe  the  procedure 
for  each  fluid. 

Pleural  Fluids. 

All  puncture  fluids  must  be  obtained  with  strict  aseptic 
precautions.  The  patient's  skin  should  be  painted  with 
iodine  solution,  and  the  needle  and  syringe  must  have  been 
boiled.  The  needle  should  be  a  fairly  long  and  stout  one, 
and  the  syringe  should  be  capable  of  holding  at  least 
10  c.c.  The  place  chosen  for  puncture  necessarily  varies 
with  the  physical  signs  present,  but  in  the  majority  of  cases 
the  puncture  is  best  made  midway  between  the  posterior 
axillary  and  scapular  lines  in  the  ninth  or  tenth  space.  If 
much  fluid  is  present  the  syringe  when  full  is  detached 
from  the  needle  and  emptied  into  a  sterile  test  tube  and  the 
process  repeated  for  a  second  tube.  The  fluid  withdrawn  may 
be  obviously  purulent  or  more  or  less  clear. 
Purulent  pleural  exudates  are  examined  as  follows  : — 
(1)  Observe  whether  they  are  offensive  or  non-offensive.  The 
majority  of  these  effusions  have  little  odour  ;  a  minority  stink 
most  evilly.  Stinking  pus  from  the  chest  is  of  diagnostic 
significance,  since  it  indicates  pretty  certainly  that  one  is  not 
dealing  with  a  simple  empyema,  that  is  with  an  abscess 
confined  to  the  pleura.  The  smell  is  produced  in  the  great 
majority  of  cases  by  a  long,  thin  saprophytic  bacillus  whose 
normal  habitat  is  the  respiratory  or  alimentary  tract.  In  a 
very  small  percentage  of  cases  the  smell  may  be  due  to,  or 
augmented  by,  the  presence  of  the  bacillus  coli  or  other 
members  of  that  group  of  organisms.  The  significance  of 
these  bacilli  is  that  a  communication  has  taken  place  between 
the  pleural  cavity  and  the  lung  or  between  the  pleura  and  an 


GENERAL   PROCEDURE— FLUIDS.  197 

abdominal  viscus.  The  presence  of  a  stinking  fluid,  therefore, 
combined  with  the  information  acquired  by  further  examina- 
tion, suggests  one  of  the  following  alternatives : — A  primary 
abscess  of  the  pleura  (or  empyema)  has  ruptured  into  the 
lung ;  a  primary  abscess  of  the  lung  has  ruptured  into 
the  pleura ;  an  abdominal  abscess  has  ruptured  through 
the  diaphragm  into  the  pleura ;  a  bronchiectatic,  and  not  a 
pleural,  abscess  has  been  punctured.  The  prognosis  for  any  of 
these  alternatives  is  evidently  less  favourable  than  for  an 
uncomplicated  empyema. 

(2)  Make  film  preparations.  Stain  with  carbol-thionin  and 
examine.  The  cells  present  will  be  found  to  consist  almost 
entirely  of  polynuclears. 

Organisms  are  usually  to  be  seen  in  addition.  The  organism 
most  commonly  met  with  is  an  extracellular  diplococcus 
with  pointed  ends,  the  pneumococcus.  The  great  majority 
of  empyemata  follow  lobar  pneumonia,  and  in  most  cases  the 
pneumococcus  is  present  in  large  numbers  and  in  pure  culture. 
The  diagnosis  of  the  causative  organism  can  be  made  with 
considerable  certainty  from  film  preparations  in  many  cases,  but 
it  is  advisable  always  to  confirm  by  cultural  tests.  Numerous 
other  organisms  may  appear  in  pairs  in  pus  films,  and  the 
pneumococcus  often  forms  quite  short  chains  of  4  to  8  members. 

The  organisms  found  next  in  order  of  frequency  are 
streptococci.  These  may  appear  in  chains  of  considerable 
length,  are  often  intracellular,  and  usually  prove  on  culture  to 
be  of  the  ordinary  S.  pyogenes  type.  Streptococcal  purulent 
effusions  are  not  infrequently  met  with  in  young  children  as  a 
sequel  of  broncho-pneumonia,  and  may  also  occur  as  part  of  a 
general  septicaemia  or  as  a  terminal  infection  in  chronic 
general  diseases.  Streptococcal  effusions  are  of  much  less 
favourable  prognosis  than  pneumococcal. 

Staphylococci  are  occasionally  found  in  these  fluids,  but 
they  are  practically  never  the  primary  cause  of  a  local 
empyema.  The  presence  of  a  staphylococcus  is  suggestive 
either  of  a  general  pyaemia  or  of  an  infection  secondary  to 
some  other  process,  most  commonly  a  tuberculous  one. 

Tubercle  bacilli  may  be  present  in  apparently  purulent 
exudates.  They  will,  of  course,  not  appear  in  the  carbol- 
thionin  preparation,  but  are  to  be  suspected  on  the  following 
grounds  : — If  the  cells  in  the  film  preparation  are  extremely 


198  CLINICAL   PATHOLOGY. 

degenerate  and  no  bacteria  are  to  be  seen  ;  if  the  cultures  on 
ordinary  media  are  subsequently  sterile  ;  if  only  staphylococci 
or  the  long,  thin  bacilli  of  a  stinking  exudate,  or  both,  are  found ; 
if  the  condition  was  a  pyo-pneurno-thorax  and  not  a  simple 
empyema;  if  there  is  other  evidence  of  tuberculosis  in  the 
patient. 

Saprophytic  bacilli. — These  are  practically  confined  to 
the  stinking  exudates,  and  are  usually  associated  with  other 
organisms.  They  may  be  very  numerous  in  the  film  pre- 
parations, and  appear  mainly  as  long,  thin  curved,  and  often 
beaded,  extracellular  bacilli.  They  are  sometimes  so  numerous 
as  to  form  a  regular  background  to  the  film.  Their  diagnostic 
import  has  been  already  referred  to.  They  are  in  all 
probability  non-pathogenic,  and  they  do  not  grow  upon  the 
ordinary  media  in  aerobic  culture. 

Influenza  bacilli  are  occasionally  met  with  in  purulent 
pleural  exudates.  They  appear  as  minute  intracellular  bacilli, 
and  should  be  proved  to  be  negative  to  Gram's  stain. 

Other  bacteria,  such  as  are  rarely  found  in  the  chest, 
include  colon  bacilli,  typhoid  bacilli,  bacillus  proteus,  bacillus 
pyocyaneus,  the  pneumobacillus. 

(3)  Identify  the  causative  organisms  by  cultural  or  other 
methods.  The  bacteria  seen  in  the  film  preparations  should 
suggest  the  further  procedure  necessary  for  their  exact 
identification.  When  pneumococci  are  suspected,  subculture 
from  the  pus  into  litmus  milk  and  on  to  agar.  In  the  case  of 
streptococci  or  staphylococci  take  the  primary  cultures  on  to 
agar  and  into  broth. 

In  suspected  tuberculous  fluids  proceed  as  follows  : — Shake 
up  about  2  c.c.  of  the  pus  with  about  10  c.c.  of  20  per  cent, 
antiformin  (page  156).  Stand,  preferably  in  a  warm  place, 
until  the  pus  is  mainly  dissolved.  Centrifuge  at  a  high  speed. 
Pour  off  the  supernatant  fluid,  and  stain  films  of  the  deposit  by 
the  Ziehl-Neelsen  method.  The  bacilli  may  be  present  in  such 
films  in  considerable  numbers ;  more  often  they  are  very  scanty, 
and  they  may  not  be  found  at  all.  If  no  bacilli  are  found  a 
portion  of  the  antiformin  residue,  after  being  washed,  should 
be  injected  into  a  guinea-pig  and  another  portion  cultivated 
upon  Dorset's  egg  medium. 

Influenza  bacilli  should  be  subcultured  on  to  nasgar,  or 
preferably  blood-agar. 


GENERAL   PROCEDURE  -FLUIDS.  199 

"    The  majority  of  other  bacteria  are  rarely  met  with.     In  the 
case  of  bacilli  of  the  colon  group  a  primary  broth  culture 
should  be  made  and  subsequently  plated  out  on  Petri  dishes. 
More    or   less    clear   pleural   exudates.— These   are 

examined  as  follows : — 

(1)  Naked-eye  appearance. — The  great  majority  of  these 
fluids  are  of  a  pale  straw  colour  and  form  an  almost  complete 
jelly-like  clot  on  standing.  The  exudates  as  a  rule  clot  more 
firmly  than  the  transudates.  The  presence  of  blood  in  any 
quantity  should  be  noted. 

(2)  The  chemical  examination. — This  need  be  by  no  means 
exhaustive,  and  unless  a  considerable  quantity  of  fluid  is  avail- 
able can  be  omitted  altogether.  The  reaction  of  these  fluids 
is  generally  alkaline.  The  specific  gravity  is  commonly  above 
1,020  in  the  case  of  exudates  and  below  1,020  for  transudates. 
The  coagulable  proteids  can  be  roughly  estimated  by  acidifying 
the  fluid  in  a  test  tube,  boiling,  and  allowing  the  precipitate  to 
settle.     The  exudates  often  become  almost  solid  on  boiling. 

(3)  The  cy  tological  examination. — This  is  of  great  importance 
and  should  never  be  omitted.  It  is  convenient  to  have  two 
test  tubes  of  the  fluid  in  order  that  one  portion  may  be  left 
uncontaminated  for  a  subsequent  bacteriological  investigation. 
If  only  one  tube  is  available  a  portion  should  be  poured  off 
into  a  second  tube  for  cyto-diagnosis. 

The  clot  must  first  be  broken  up,  and  this  can  be  done  either 
by  shaking  the  tube  or  stirring  up  the  contents  with  a  glass 
rod.  The  fluid  is  then  centrifuged  at  a  moderate  speed, 
preferably  on  a  water  or  electrically  driven  centrifuge,  but 
a  hand  centrifuge  will  serve  the  purpose.  All  centrifuge 
tubes  should  be  carefully  rinsed  out  with  distilled  water 
before  use. 

After  centrifuging  empty  out  the  supernatant  fluid  by 
simply  turning  the  tube  upside  down.  The  last  drop,  con- 
taining the  majority  of  the  cells,  will  remain  in  the  bottom  of 
the  tube.  (It  is  a  wise  precaution  to  preserve  the  supernatant 
fluid,  if  there  is  not  a  large  quantity  available.) 

Wrap  a  very  small  piece  of  absorbent  cotton  wool  around 
the  tips  of  a  small  pair  of  fine-pointed  forceps.  Soak  the 
wool  in  the  centrifuged  deposit  and  make  moderately  thick 
films  in  the  centres  of  2  slides.  Stain  one  slide  with  carbol- 
thionin,  the  other  with  Leishman's  stain. 


200  CLINICAL   PATHOLOGY. 

The  Leishrnan-stained  film  should  be  treated  in  the  same 
manner  as  a  blood  film,  but  given  the  following  times :  — 
Leishman  alone,  ^  minute. 

Leishman  -4-  2  volumes  of  distilled  water,  5  minutes. 
Distilled  water  only,  2  minutes. 

Examine  the  films  with  an  oil  immersion  lens.  In  the 
majority  of  these  pleural  fluids  the  cells  are  small  lymphocytes  ; 
in  the  minority  either  polynuclear  neutrophils  or  endothelial 
cells  predominate. 

The  lymphocytes  are  often  very  numerous,  and  20  or 
30  are  commonly  seen  in  one  field  of  a  j^th  inch  objective. 
They  often  form  at  least  80  per  cent,  of  the  cells  present,  the 
remainder  consisting  of  endothelials,  and  large  lymphocytes, 
with  occasional  polynuclear  or  other  cells. 

Such  a  lymphocytic  effusion  is  almost  certain  evidence  of 
a  tuberculous  affection,  syphilitic  disease  of  the  pleura  being 
practically  unknown.  Very  rarely  a  lymphocytic  exudate  may 
occur  with  pleural  or  mediastinal  neoplasms. 

A  deposit  of  endothelial  cells  occurs  most  frequently  in 
cardiac  or  renal  dropsy.  They  may  also  be  found  with 
malignant  growths. 

Polynuclear  neutrophils  may  predominate  in  an  apparently 
clear  or  in  a  slightly  turbid  fluid.  They  have  the  same 
significance  as  in  an  obviously  purulent  fluid. 

Eosinophil  cells  are  occasionally  found  in  the  clear  fluid 
which  exceptionally  follows  a  lobar  pneumonia,  and  still  more 
rarely  in  a  tuberculous  exudate.  When  present  in  excess 
a  Irydatid  cyst  should  always  be  suspected  and  the  fluid 
examined  accordingly  (page  209). 

(4)  The  bacteriological  examination. — This  varies  with  the 
type  of  cell  present. 

In  the  case  of  a  lymphocytic  exudate  proceed  as  follows  : — 
Fill  2  centrifuge  tubes  (of  about  10  c.c.  capacity  each)  two- 
thirds  full  of  the  fluid.  Add  one- third  of  absolute  alcohol. 
Invert  several  times  and  stand  for  5  minutes.  A  copious 
precipitate  forms.  Centrifuge  at  a  high  speed  for  10  minutes. 
Pour  off  the  supernatant  fluid.  If  the  deposit  is  comparatively 
small  in  amount  make  thick  films  from  it  and  stain  for 
tubercle  bacilli  in  the  ordinary  way.  If  the  deposit  is  very 
bulky  (as  is  usually  the  case)  fill  the  tubes  with  20  per  cent, 
antiformin.     Shake   thoroughly  and  stand  in  a  warm   place 


GENERAL   PROCEDURE— FLUIDS.  201 

until  the  precipitate  is  almost  dissolved.  Centrifuge  again  at 
a  high  speed  for  10  minutes.  Make  films  of  the  deposit  and 
stain  for  tubercle  bacilli.  A  prolonged  search  is  necessary  to 
demonstrate  the  bacilli,  and  in  the  case  of  the  majority  of 
pleural  fluids  of  tuberculous  origin  no  bacilli  can  be  found. 
In  cases  of  exceptional  importance  the  antiformin  deposit 
should  be  washed  two  or  three  times  with  sterile  saline  and 
one  portion  rubbed  over  the  surface  of  Dorset's  egg  medium, 
the  other  portion  injected  into  the  leg  of  a  guinea-pig.  Prefer- 
ably the  centrifuged  deposit  of  the  original  fluid  is  injected, 
without  other  treatment,  into  a  guinea-pig. 

Where  endothelial  cells  are  in  excess  no  bacteriological 
examination  is  necessary. 

With  an  excess  of  polynuclear  neutrophils  the  procedure  is 
the  same  as  for  purulent  exudates,  except  that  it  is  advisable 
to  add  the  fluid  in  greater  bulk  to  the  media.  As  an  alter- 
native the  fluid  may  be  centrifuged  in  sterile  tubes  and  the 
deposit  of  pus  used  for  inoculation  of  the  media. 

Lung  puncture. — It  occasionally  happens  that  a  chest 
deemed  to  contain  fluid  proves  on  exploratory  puncture  to  be 
filled  with  solid  lung.  On  such  occasions  a  small  quantity  of 
blood-stained  fluid  is  removed  in  the  needle  and  should  be 
reserved  for  film  and  culture  preparations.  The  pneumococcus 
or  other  organisms  can  often  be  identified  by  these  means,  and 
a  diagnosis  of  the  condition  made. 

Lung  puncture  may  be  performed  deliberately  as  a  means 
of  diagnosis,  but  the  procedure  can  hardly  be  said  to  be 
entirely  free  from  risk,  and  should  be  avoided  as  a  routine 
method. 

Pericardial  Fluids. 

Puncture  of  the  pericardium  is  rarely  performed.  It  is 
advised  to  make  the  puncture  with  a  fine  needle  in  the  fourth 
left  intercostal  space  half  an  inch  from  the  edge  of  the 
sternum.  The  fluid  withdrawn  should  be  examined  in  exactly 
the  same  way  as  a  pleural  fluid  both  by  cytological  and 
bacteriological  methods.  Those  fluids  for  which  pericardial 
puncture  is  performed  are  usually  purulent  and  frequently 
associated  with  a  purulent  pleurisy. 


CHAPTEE  XV. 

peritoneal  fluids — cerebrospinal  fluids — synovial 
fluids cysts,  etc. 

Peritoneal  Fluids. 

Before  puncturing  the  peritoneal  cavity  the  bladder  must 
be  emptied.  The  puncture  is  best  made  in  the  middle  line 
and  midway  between  the  umbilicus  and  the  pubes. 

The  fluid  is  examined  in  much  the  same  way  as  a  pleural 
fluid. 

Purulent  peritoneal  fluids  are  rarely  withdrawn  by 
puncture,  being  more  commonly  obtained  during  a  laparotomy 
for  general  peritonitis.  The  organisms  present  in  such  fluids 
usually  include  the  bacillus  coli.  Cocci  are  often  seen  in 
addition.  Streptococci  in  pure  culture  are  of  the  worst  pos- 
sible prognosis.  Pure  pneumococcal  exudates  are  more  fre- 
quent in  young  children,  are  as  a  rule  associated  with 
comparatively  slight  constitutional  disturbance,  and  are  of 
fairly  good  prognosis.  B.  pyocyaneus  is  occasionally  met 
with,  and  is  usually  associated  with  a  particularly  virulent 
peritonitis.  Purulent  fluids  are  sometimes  obtained  in  associa- 
tion with  the  tubercle  bacillus,  a  secondary  infection  with 
the  B.  coli,  or  other  intestinal  organisms  having  taken  place. 
Almost  all  of  the  pyogenic  organisms  are  found  from  time 
to  time  in  the  peritoneal  cavity,  and  the  examination  of 
purulent  peritoneal  fluids  is  conducted  on  exactly  the  same 
lines  as  those  already  laid  down  for  the  examination  of  "  pus  r' 
generally. 

Clear  peritoneal  fluids  are  examined  in  the  same  way  as 
clear  pleural  fluids. 

Lymphocytic  fluids  indicate  a  tuberculous  lesion ;  endothelial 
fluids  are  associated  with  cardiac  or  renal  dropsy,  with  cirrhosis 
of  the  liver,  with  malignant  disease,  or  with  any  abdominal 
lesion  which  tends  to  obstruct  the  portal  circulation. 

Polynuclear  neutrophils  may  be  present  in  apparently  clear 
peritoneal  fluids  and  in  association  with  the  ordinary  pyogenic 


PEEITONEAL   FLUIDS— CYSTS.  203 

organisms.  With  such  fluids  ifc  is  always  wise  to  examine  films 
of  the  deposit  for  tubercle  bacilli,  otherwise  the  underlying 
cause  of  the  condition  may  be  missed.  The  tubercle  bacilli 
may  be  present  in  considerable  numbers. 

The  cytology  of  peritoneal  fluids  is  for  some  cause  not  quite 
so  satisfactory  as  that  of  pleural  fluids.  The  cellular  deposit 
is  very  frequently  a  mixed  one,  and  the  diagnostic  value  of  the 
findings  is  not  very  great  unless  the  predominant  cell  is  present 
in  overwhelming  numbers. 

Lymphocytic  fluids  should  be  examined  for  tubercle  bacilli 
in  the  same  way  as  pleural  fluids.  Cultures  should  be  made 
of  the  acute  inflammatory  or  polynuclear-containing  fluids. 

Opalescent  fluids  are  occasionally  withdrawn  from  the 
peritoneal  cavity,  and  still  more  rarely  from  the  pleural  sacs. 
The  opalescence  is  not  removed  by  filtration,  nor  by  centri- 
fuging,  nor  by  extraction  with  ether,  and  is  apparently  due  to 
the  presence  of  a  lipoid  body  combined  with  a  proteid.  The 
significance  of  these  striking-looking  fluids  is  not  known,  but 
they  seem  to  be  most  frequently  associated  with  the  dropsy  of 
parenchymatous  nephritis. 

Extremely  rare  are  genuine  chylous  fluids,  in  which  the 
opalescence  is  due  to  actual  fat  and  is  removed  by  extraction 
with  ether.  Such  fluids  are  associated  with  lesions  of  the 
thoracic  duct,  and  occur  in  filariasis.  They  are  to  be  distin- 
tinguished  from  the  pseudochylous  fluids  referred  to  above. 

Cbrebro-spinal  Fluid. 

The  fluid  is  obtained  by  puncture  through  the  fourth  lumbar 
space,  a  special  hollow  needle  provided  with  a  stilette  being 
required  for  the  operation.  Needle  and  stilette  should  be 
made  of  flexible  drawn  nickel  and  of  a  length  of  3J  inches. 
Do  not  use  an  ordinary  steel  exploring  needle,  since  it  is  liable 
to  break  in  the  tissues.  An  all-glass  syringe  should  be  fitted  to 
the  needle,  but  it  is  advisable  so  far  as  possible  to  avoid  strong 
suction  with  it. 

The  patient  is  placed  in  the  left  lateral  position  with  the 
back  well  bent  and  the  knees  drawn  up  so  as  to  approximate 
to  the  chin.  A  line  is  drawn  across  the  back  to  join  the 
highest  points  of  the  iliac  crests,  and  the  interspinous  space 
immediately  below  this  line  is  defined  with  the  tip  of   the 


204  CLINICAL   PATHOLOGY. 

finger.  An  area  of  skin  is  painted  with  iodine,  and  may 
be  anaesthetised  with  the  ethyl-chloride  spray.  The  needle 
is  plunged  firmly  into  the  interspinous  space  one-fourth  of 
an  inch  to  one  side  of  the  middle  line  and  pushed  steadily 
onwards  and  directly  forwards  until  the  canal  is  reached.  In 
the  majority  of  adult  patients  the  needle  has  to  be  passed 
almost  up  to  the  hilt,  and  the  correct  position  of  the  point  is 
recognised  by  the  sudden  and  easy  passage  of  the  needle  just 
as  the  canal  is  reached.  The  patient  may  also  complain  of  a 
sensation  of  pain  or  tingling  in  the  leg.  On  withdrawing  the 
stilette  the  fluid  may  gush  out,  or,  as  is  more  frequent,  escape 
drop  by  drop.  The  flow  can  sometimes  be  started  by  cautiously 
applying  suction  with  the  syringe. 

The  fluid  is  collected  in  clean  sterile  test  tubes,  and  if  the 
first  sample  is  tinged  with  blood  a  second  tube  should  be  ready 
as  soon  as  the  fluid  runs  clear.  It  is  important  to  obtain  a 
sample  free  from  blood.  About  10  c.c.  of  fluid  can  be  removed 
with  impunity. 

After  withdrawing  the  trocar  seal  the  puncture  with  collodion, 
and  direct  that  the  patient  be  kept  in  bed  with  the  head  low 
for  a  period  of  24  hours. 

The  fluid  is  examined  as  follows  : — 

(1)  Naked-eye  examination. — The  normal  cerebro-spinal 
fluid  is  a  clear,  limpid  fluid  resembling  water.  In  disease 
small  flocculent  threads  may  be  seen  floating  in  the  fluid,  an 
appearance  particularly  common  in  syphilitic  and  parasyphilitic 
conditions.  Sometimes  on  standing  a  delicate  clot,  having  the 
appearance  of  a  fine  cobweb,  slowly  forms  in  the  tube.  This 
formation  of  a  clot  is  certain  evidence  of  disease,  and  suggests 
either  tuberculous  or  meningococcal  meningitis.  In  suppura- 
tive meningitis,  whether  due  to  the  meningococcus  or  to  other 
pyogenic  organisms  such  as  the  pneumococcus,  every  grade  of 
turbid  fluid  is  found,  from  a  mere  cloudiness  to  actual  pus. 
The  presence  of  blood,  if  bright  in  colour,  is  usually  due  to  the 
puncture  of  a  vessel ;  if  dark,  intimately  mixed  with  the  fluid 
and  persisting  during  the  whole  time  the  fluid  is  running,  it  is 
suggestive  of  intradural  haemorrhage,  such  as  occurs  in  fracture 
of  the  base  of  the  skull. 

(2)  Cytological  and  bacteriological  examinations. — 
If  normal  spinal  fluid  is  centrifuged  for  10  minutes  at  a 
moderate  speed,  if  the  tube  is  emptied  by  inversion  and  a 


PEEITONEAL   FLUIDS— CYSTS,   ETC.  205 

fairly  thick  film  the  size  of  a  sixpence  is  made  with  a  cotton-wool 
swab  in  the  manner  described  for  pleural  fluids,  practically  no 
cells  are  found  in  the  film.  The  presence  of  more  than  2  or 
3  cells  to  a  film  in  preparations  made  by  this  method  is 
evidence  of  disease.  It  often  happens  in  disease  that  no 
obvious  sediment  is  found  after  centrifuging ;  the  last  drop 
may  nevertheless  be  rich  in  cells.  The  cells  present  in  these 
fluids  are  practically  of  only  two  kinds — small  lymphocytes  and 
polynuclear  neutrophils  ;  endothelial  cells  in  excess  are  never 
seen.  If  small  lymphocytes  are  present  the  fluid  is  either 
syphilitic  or  tuberculous. 

If  a  syphilitic  lesion  is  suspected  a  Wassermann  reaction 
must  be  performed  with  the  fluid  and  with  the  patient's  serum. 
In  syphilis  of  the  central  nervous  system  the  reaction  is 
typically  negative  in  the  fluid  and  positive  in  the  serum. 
In  tabes  and  general  paralysis  the  reaction  is  usually  positive 
in  both  serum  and  fluid. 

If  a  tuberculous  lesion  is  suspected  proceed  as  follows : — 

Half-fill  two  centrifuge  tubes  with  the  fluid,  and  add  to  each 
an  equal  volume  of  absolute  alcohol.  Mix  thoroughly  and  stand 
for  a  few  minutes.  In  the  case  of  normal  fluids  practically  no 
turbidity  results  ;  in  meningeal  disease,  whether  tuberculous, 
syphilitic,  or  suppurative,  a  more  or  less  marked  opalescence 
forms  in  the  fluid,  owing  to  the  precipitation  of  proteids.  This 
stage  of  the  proceedings  therefore  yields  confirmatory  evidence 
of  the  cytological  findings.  The  tubes  are  then  centrifuged  at 
as  high  a  speed  as  possible  for  about  10  minutes.  In  the 
majority  of  cases  a  small  but  obvious  sediment  is  found  at  the 
bottom  of  the  tubes.  As  thick  films  as  possible  are  made  from 
the  sediment  and  stained  for  tubercle  bacilli  in  the  ordinary 
way.  Bacilli  may  be  present  in  considerable  numbers  ;  more 
commonly  they  are  very  scanty ;  a  careful  search,  however, 
should  reveal  the  bacilli  in  at  least  70  per  cent,  of  the  cases. 

If  polynuclear  cells  are  found  some  form  of  septic  meningitis 
is  present.  The  causative  organism  should  be  sought  for  in 
the  films.  The  meningococci  are  seen  as  Gram-negative 
diplococci,  the  majority  of  which  are  within  the  polynuclear 
cells ;  but  a  number  of  extracellular  cocci  are  nearly  always 
present  as  well.  In  meningococcal  exudates  the  first  cultures 
should  be  made  on  nasgar  and  in  milk,  and  in  a  certain 
number  of  cases,  even  when  the  organisms  are  abundant  in  the 


206  CLINICAL   PATHOLOGY. 

films,  the  cultural  results  are  negative.  Once  a  growth  has 
been  obtained  the  sub-cultures  grow  readily,  provided  they  are 
made  at  frequent  intervals.  Pneumococci  are  sometimes 
found  in  the  spinal  fluid,  and  are  recognised  as  Gram-positive, 
lanceolate,  extracellular  diplococci.  The  cultures  should  be 
made  in  milk  and  on  agar.  The  pneumococcal  cases  run  a 
rapidly  fatal  course  of  1  to  3  days  as  a  rule  ;  consequently 
the  prognosis  is  far  less  favourable  than  in  the  meningococcal 
infections,  which  run  a  more  chronic  course  with  a  fairly  high 
percentage  of  recoveries.  Other  organisms  which  may  be  found 
in  the  cerebro-spinal  fluid  include  streptococci,  influenza  bacilli 
and  colon  bacilli.  Diphtheroid  bacilli  and  staphylococci  are 
occasionally  grown  in  culture,  but  are  usually  to  be  regarded 
as  skin  contaminations. 

To  recapitulate  the  findings  of  a  spinal  fluid  deposit. 
Absence  of  cells  is  very  strong  evidence  against  any  variety 
of  meningitis.  The  normal  fluid  examined  in  the  manner 
described  here  yields  no  cells  in  the  film  preparation.  Small 
lymphocytes  mean  syphilis  or  tuberculosis.  Polynuclears 
mean  septic  meningitis,  of  which  the  most  common  variety  is 
due  to  the  meningococcus,  the  next  common  to  the  pneumo- 
coccus. 

The  examination  of  the  cellular  deposit  may  be  obscured  by 
the  presence  of  blood,  and  it  must  also  be  recognised  that  the 
preponderance  of  the  cells,  whether  lymphocytes  or  polynuclears, 
is  a  matter  of  degree.  A  few  polynuclears  will  be  found 
in  a  tuberculous  or  syphilitic  meningitis.  Lymphocytes  are 
fairly  numerous  in  meningococcal  infections,  and  particularly 
in  the  more  chronic  cases.  A  positive  interpretation  of  the 
film  requires  that  the  percentage  of  the  predominant  cell 
should  be  at  least  70. 

(3)  The  chemical  examination. — If  10  c.c.  of  fluid  have 
been  withdrawn  a  few  simple  chemical  tests  should  be  per- 
formed, since  these  are  confirmatory  of  previous  findings. 

The  globulin  content  of  the  fluid  is  a  guide  to  the  presence 
of  meningeal  inflammation,  whether  tuberculous,  syphilitic, 
or  septic.  It  can  be  investigated  by  the  method  of  Nonne  and 
Apelt.  A  saturated  solution  of  ammonium  sulphate  is  prepared 
as  follows  : — 85  grammes  of  the  pure  salt  (Merck)  are  boiled  in 
a  flask  with  100  c.c.  of  distilled  water  until  no  more  salt  is 
taken  up.      The  solution  is  allowed  to  cool  and  is  filtered. 


PEEITONEAL   FLUIDS— CYSTS,   ETC.  207 

Equal  parts  of  the  solution  and  of  the  fluid  are  mixed  and 
allowed  to  stand  at  room  temperature  for  3  minutes. 
After  3  minutes  note — 

(1)  Opacity; 

(2)  Opalescence ; 

(3)  Slight  opalescence  ; 

(4)  Trace  of  opalescence. 
1,  2,  and  3  are  positive ;  4  is  negative. 

The  spinal  fluid  must  not  be  heated  and  must  contain  no 
blood.  Very  similar  readings  are  obtained  by  the  simple 
addition  of  absolute  alcohol,  as  described  under  the  method 
for  demonstrating  tubercle  bacilli  in  tbe  fluid. 

The  spinal  fluid  should  also  be  examined  by  the  ordinary 
Fehling  test  for  the  presence  of  a  reducing  substance.  A  trace 
of  sugar  is  present  in  the  normal  fluid  ;  it  is  usually  absent  in 
cases  of  meningitis. 

Synovial  Fluids. 

The  puncture  of  joints  for  diagnostic  purposes  should  only 
be  undertaken  in  ideal  surroundings  and  with  the  strictest 
aseptic  precautions.  Provided  there  is  a  large  excess  of  fluid 
in  one  of  the  large  joints  and  every  care  is  taken,  there  is  no 
real  risk  in  the  procedure.  The  point  at  which  to  make  the 
puncture  necessarily  varies  for  each  joint,  but  no  difficulty 
should  be  experienced  if  the  needle  is  plunged  directly  through 
the  skin  into  the  most  obviously  distended  portion  of  the  joint 
cavity.  The  needle  should  be  of  sharp  steel  fitted  with  an  all- 
glass  syringe  and  with  a  stilette.  After  withdrawal  of  the 
needle  the  puncture  wound  must  be  sealed  with  collodion. 

The  removal  of  the  fluid  from  a  joint  has  further  advan- 
tages. The  relief  of  tension  when  a  moderate  quantity  of 
fluid  is  withdrawn  is  in  many  cases  beneficial,  and  the  subse- 
quent preparation  of  an  autogenous  vaccine  may  be  made  use 
of  as  an  aid  to  treatment. 

In  the  puncture  of  a  distended  knee  joint  an  unexpectedly 
small  quantity  of  fluid  is  commonly  withdrawn  owing  to  the 
blocking  of  the  needle  with  synovial  fringes.  By  altering  the 
position  of  the  needle  a  further  flow  is  often  obtained. 

The  normal  synovial  fluid  is  a  sticky  viscous  material  of 
unmistakable  character.     In  disease  it  may  be  not  obviously 


208  CLINICAL   PATHOLOGY. 

altered   in   appearance,    or   it   may   be  turbid,  or  it  may  be 
obviously  purulent. 

In  all  cases  film  preparations  should  be  made  without 
centrifuging.  In  practically  every  variety  of  arthritic  disease 
the  predominant  cell  is  a  polynuclear  neutrophil,  while 
lymphocytic  and  endothelial  cells  are  usually  present  in 
addition.  The  polynuclear  cell  is  predominant,  not  only  in 
acute  lesions  due  to  gonococci  or  other  pyogenic  organisms,  but 
also  in  the  chronic  distended  joints  of  rheumatoid  arthritis. 
Lymphocytes  may  be  more  numerous  in  acute  tuberculous 
joints,  but  the  cytology  of  synovial  fluids  is  not  of  striking 
diagnostic  value.  The  cells  in  carbol-thionin  preparations  are 
seen  lying  on  a  deeply-stained  background  of  the  synovial  fluid. 

All  film  preparations  should  be  carefully  searched  for 
bacteria,  and  these  should  be  seen  in  all  the  acute  septic  cases 
due  to  streptococci,  pneumococci,  or  staphylococci,  and  in 
many  of  the  gonorrhceal  cases  if  a  very  careful  and  prolonged 
search  is  made.  In  these  affections  the  findings  of  the  film 
preparations  must  always  be  confirmed  by  cultural  methods. 

In  tuberculous  cases  no  organisms  are  seen  in  the  carbol- 
thionin  films  unless  a  secondary  infection  has  taken  place. 
The  material  withdrawn  in  such  cases  is  often  suggestively 
caseous,  and  the  cells  are  necrotic  and  many  of  them 
unrecognisable.  A  portion  of  the  fluid  should  be  treated  with 
antiformin,  centrifuged  and  examined  for  tubercle  bacilli  in 
the  ordinary  way.     The  bacilli  may  be  fairly  numerous. 

In  the  large  group  of  arthritic  affections  known  as  "  rheu- 
matoid "  no  organisms  are  seen  in  the  films  and  none  are 
grown  in  any  culture  media.  Paracentesis  of  acute  rheumatic 
joints  is  rarely  performed,  partly  because  the  effusion  is  so 
often  slight  in  amount  and  is  mainly  periarticular.  A  short- 
chained  streptococcus  has  been  recovered  in  these  cases,  and 
is  by  some  considered  to  be  the  cause  of  rheumatic  fever.  A 
similar  coccus  has  been  found  in  almost  every  rheumatic  lesion, 
including  the  angina,  the  nodules,  the  valvular  vegetations, 
and,  in  cases  of  infective  endocarditis,  in  the  circulating  blood. 
The  etiology  of  rheumatic  fever  is,  however,  still  sub  judice. 

Hydrocele  and  spermatocele  fluids.  —  These  are 
obtained  by  puncture  through  the  tensest  portion  of  the  cysts, 
care  being  taken  to  avoid  wounding  one  of  the  scrotal  vessels. 
Hydrocele  fluid  is  commonly  clear  and  straw-coloured.      On 


PEKITONEAL   FLUIDS— CYSTS.  209 

standing  a  quantity  of  cholesterin  crystals  separates  out. 
The  cells  present  are  mainly  endothelial,  and  no  organisms 
are  found.  In  cases  where  secondary  infection  has  occurred 
polynuclear  neutrophils  predominate,  and  the  ordinary 
pyogenic  organisms,  most  commonly  staphylococci,  are 
obtained.  Spermatocele  fluid  is  usually  milky  in  appearance, 
and  in  almost  all  cases  large  numbers  of  spermatozoa  are 
seen  under  the  microscope.  The  spermatozoa  are  best 
examined  unstained,  a  drop  of  the  fluid  being  placed  on  a  slide 
with  a  cover-slip  over  it  and  watched  under  the  ^-inch  objective. 
Stained  preparations  of  dried  films  can  be  made  with  carbol- 
thionin  or  Leishman's  stain. 

Cysts. 

For  all  cyst  fluids  a  chemical  and  a  microscopical  exami- 
nation are  required.  Bacteriological  investigations  are  rarely 
called  for.  In  the  majority  of  cases  the  nature  of  the  cyst  is 
clearly  determined  on  clinical  grounds  or  at  operation,  and 
the  laboratory  investigations  are  for  purposes  of  confirmation. 
The  examinations  made  should  naturally  be  those  most  appro- 
priate to  the  type  of  cyst  suspected.  When  the  nature  of  the 
cyst  is  quite  obscure  the  following  routine  procedure  should 
be  adopted.  Note  the  amount  and  naked-eye  appearance  of 
the  fluid.  Test  the  reaction  and  the  specific  gravity.  Esti- 
mate roughly  the  amount  of  coagulable  proteid.  Test  for  the 
presence  and  amount  of  urea.  Test  for  the  presence  of  a 
reducing  substance.  Estimate  roughly  the  amount  of 
chlorides  present.  Examine  the  centrifuged  deposit  in  fresh 
and  stained  specimens,  as  to  the  nature  of  the  cells,  and  the 
presence  of  crystals  or  of  hydatid  hooklets.  If  there  is  reason 
to  suspect  a  pancreatic  cyst,  examine  also  for  the  presence  of 
ferments.  Prepare  paraffin  sections  of  the  cyst  wall,  if  available. 
The  following  is  a  brief  description  of  the  more  important 
cyst  fluids  :  — 
Hydatid  cysts. 

Specific  gravity  about  1,010. 

Proteids  very  scanty  or  absent. 

Chlorides  abundant. 

Sugar  occasionally  present. 

Urea  present  in  very  small  amount. 

Hydatid  hooklets  in  centrifuged  deposit. 

14 


210  CLINICAL   PATHOLOGY. 

The  really  diagnostic  feature  is  the  presence  of  hooklets. 

The   hooklets   are   sharply   pointed   at  one  end  and  barbed. 

Occasionally  a  well-formed  scolex  with  a  circle  of  hooklets  may 

be  found  (page  334). 

Pancreatic  cysts. 

Specific  gravity,  1,010—1,020. 

Keaction  alkaline. 

Proteids  in  varying  amounts. 

Chlorides  usually  scanty. 

Urea  often  a  trace. 

Cholesterin  usually  present. 

Trypsin,  lipase,  and  diastase  present. 

The    characteristic    constituents   of    these    cysts    are   the 

ferments,  which  can  be  found  in  the  majority  of  cases.     They 

are  tested  for  as  follows  : — 

Trypsin. — Put  in  one  test  tube  about  5  c.c.  of  the  fluid  ;  in  a 

second  test  tube  5  c.c.  of  the  fluid  and  bring  to  the  boiling 

N 
point ;   in  a  third  test  tube  4  c.c.  of  the  fluid,  and  1  c.c.  of  — 

N 
NaOH ;  in  a  fourth  tube  5  c.c.  of  ^  NaOH.     Label  each  tube. 

Add  to  each  tube  veiy  thin  strips  of  hard-boiled  white  of  egg. 
Cork  the  tubes  with  wool  plugs,  place  in  the  incubator  at 
37°  C.  and  examine  at  the  end  of  1  hour,  2  hours,  and  12 
hours.  Definite  digestion  of  the  proteid  should  have  occurred 
in  tubes  1  and  3  at  the  end  of  2  hours,  and  digestion  should 
be  complete  the  next  morning. 

Lipase. — Exactly  neutralise  the  fluid  with  dilute  HC1.  Add 
a  trace  of  calcium  chloride.  Add  a  few  drops  of  ethyl  butyrate 
in  a  test  tube  and  a  few  drops  of  litmus  solution.  Put  up  a 
control  tube  of  the  boiled  fluid.  Incubate  for  12  hours  at 
37°  C.     The  litmus  is  turned  red  if  lipase  is  present. 

Diastase. — Add  a  small  amount  of  starch  emulsion  to  the 
fluid.  Incubate  and  examine  at  the  end  of  1,  2,  and  12  hours. 
Put  up  control  tubes.  A  drop  of  the  starch  and  fluid  mixture  is 
taken  out  and  mixed  with  a  drop  of  iodine  solution.  When 
diastase  is  present  the  blue  coloration  is  lost  and  replaced, 
first  by  a  red  colour,  finally  by  an  absence  of  colour. 

Renal  cysts.  --Renal  cysts  proper  may  arise  from  a 
hypernephroma,  from  a  congenital  cystic  kidney,  or  from  a 
contracted  granular  or  cardiac  kidney.     Rare  single  cysts  of 


PERITONEAL   FLUIDS— CYSTS.  211 

considerable  size  may  be  found,  arising,  as  a  rule,  from  the 
lower  pole  of  an  apparently  healthy  kidney.  The  composition 
of  such  cysts  is  very  variable,  and  both  urea  and  uric  acid 
may  be  absent. 

The  contents  of  a  hydronephrosis  have  more  obviously  the 
composition  of  urine,  but  the  specific  gravity  is  usually  below 
1,010 ;  albumin  is  frequently  small  in  amount.  The  reaction 
may  be  acid,  a  point  which  immediately  suggests  a  renal 
origin.  The  most  important  constituent  is  urea,  which  may 
amount  to  from  0"4  to  l'O  per  cent.  It  must  be  remembered, 
however,  that  many  cystic  fluids  contain  a  trace  of  urea,  though 
a  percentage  of  more  than  0*2  is  very  suggestive  of  a  renal 
origin  and  more  than  0*5  is  practically  diagnostic. 

Ovarian  cysts. — The  source  of  the  fluid  may  sometimes 
be  recognised  from  the  character  of  the  cellular  deposit.  The 
cells  in  some  cases  may  be  indistinguishable  from  the 
endothelial  cells  of  a  peritoneal  fluid ;  in  other  cases  columnar 
epithelial,  or  even  ciliated,  cells  may  be  found.  Cholesterin 
crystals  are  common  in  these  fluids,  but  may  occur  in  almost 
any  variety  of  cyst. 

Other  abdominal  cysts. — These  include  mesenteric, 
retroperitoneal,  and  omental  cysts.  All  of  these  are  rare,  but 
they  may  be  of  considerable  size.  The  fluid  contents  present 
no  particularly  characteristic  feature. 

Dermoid  cysts  may  be  recognised  by  their  contents  and 
by  the  nature  of  the  cyst  wall.  The  contents  may  include  any 
of  the  skin  appendages,  such  as  bones,  teeth,  or  hair,  the  last 
being  the  most  frequent.  The  cyst  wall  consists  of  true  skin, 
and  consequently  in  a  microscopic  section  all  the  layers  of  the 
true  skin  are  found,  including  the  stratum  lucidum  and  the 
stratum  granulosum.  These  cysts  have  to  be  distinguished  from 
sebaceous  cysts,  which  contain  only  a  pultaceous  material* 
consisting  of  fatty  bodies,  cholesterin,  and  debris.  Sebaceous 
cysts,  in  common  with  thyro-glossal  and  other  cysts  of  epiblastic 
origin,  have  an  epidermal  lining.  Some  thyro-glossal  cysts  are 
lined  with  a  columnar  ciliated  epithelium. 


14—2 


SECTION   IV. 

THE   URINE. 

CHAPTEE  XVI. 

Routine  Examination — Variations  in  Amount  —Variations  in  Appearance. 

CHAPTER  XVII. 

Variations  in  Acidity,  and  Acidosis — Variations  in  Specific  Gravity — Urea 
— Proteids — Carbohydrates. 

CHAPTER  XVIII. 

Urinary  Deposits — Urinary  Calculi. 

CHAPTER  XIX. 

Special  Investigations  of  the  Urine — Bacteriology  of  the  Urino-genital 
Tract. 


CHAPTER  XVI. 

ROUTINE    EXAMINATION VARIATIONS     IN     AMOUNT — VARIATIONS    IN 

APPEARANCE. 

An  examination  of  the  urine  forms  a  part  of  the  routine 
investigation  of  every  patient.  For  the  purposes  of  an 
ordinary  clinical  examination  a  specimen  of  urine  passed  at 
the  time  can  be  made  use  of,  but  in  the  case  of  patients  con- 
fined to  bed  a  sample  taken  from  the  total  urine  passed  in 
the  24  hours  is  to  be  preferred.  For  special  purposes  the 
urine  must  be  withdrawn  by  catheter  ;  and  a  catheter  specimen 
is  necessary  for  bacteriological  investigation  other  than  that 
for  the  presence  of  tubercle  bacilli :  the  presence  of  pyogenic 
cocci  or  bacilli  in  an  ordinary  specimen  of  urine  has  no 
significance  whatever.  For  the  detection  of  small  traces  of 
albumin  or  of  pus  in  the  urine  of  women  a  catheter  specimen 
is  similarly  essential,  since  a  sample  of  urine  passed  in  the 
natural  manner  is  very  frequently  contaminated  by  leucocytes 
and  the  albuminous  secretion  of  the  vagina.  Further,  the 
comparatively  recent  advances  of  skilled  surgery  permit,  in 
special  cases,  of  the  examination  of  the  secretions  of  each 
kidney  by  means  of  ureteric  catheterisation.  The  routine 
examination  of  an  ordinary  specimen  of  urine  should  always 
be  conducted  according  to  a  settled  scheme  of  investigation, 
otherwise  in  an  important  minority  of  cases  some  point 
essential  to  the  appropriate  treatment  will  be  missed.  The 
simple  tests  described  below  must  be  applied  in  every  case, 
whether  there  is  any  reason  to  suspect  any  abnormal  sub- 
stance in  the  urine  or  not.  The  urine  may  be  found  loaded 
with  albumin  or  sugar  in  the  case  of  apparently  healthy 
persons  who  are  seeking  advice  as  to  the  necessity  of  some 
operation  that  may  well  be  postponed,  but  which  would  be 
advisable  for  normal  persons.  The  unexpected  detection  of 
pus  in  the  urine  may  turn  the  diagnosis  of  a  supposed  splenic 
enlargement  into  that  of  a  left  pyonephrosis,  and  numerous 
similar  instances  of  the  importance  of  routine  examination 
could  readily  be  adduced. 


214  CLINICAL   PATHOLOGY. 

The  Boutine  Examination  of  the  Urine. 

(1)  The  amount  passed. — The  measurement  of  the  urine 
passed  in  the  24  hours  may  be  omitted  if  no  alteration 
in  the  urinary  secretion  is  to  be  expected  and  nothing  abnormal 
is  detected  by  the  ordinary  tests.  In  any  case  the  amount  of 
urine  necessarily  varies  considerably  according  to  the  quantity 
of  fluid  taken  and  the  amount  lost  by  the  skin.  The  average 
output  of  urine  by  the  healthy  adult  male  in  the  24  hours  is 
from  40  to  50  ounces,  the  majority  of  which  is  excreted  during 
the  daytime. 

(2)  The  naked-eye  appearance. — The  urine  directly 
after  being  passed  should  be  perfectly  clear  and  of  an  amber 
colour.  The  colour  is  mainly  due  to  the  pigment  urochrome. 
While  the  naked-eye  inspection  of  a  sample  of  urine  is  of 
value  in  leading  to  the  detection  of  many  gross  pathological 
changes,  confirmatory  tests  should  always  be  employed,  since 
it  is  often  impossible  to  distinguish  by  the  eye  numerous  sub- 
stances of  similar  appearance  and  widely  different  nature. 
The  certainty  of  an  observer's  naked-eye  diagnosis  usually 
varies  inversely  with  the  carefulness  of  his  routine  examination. 

(3)  The  reaction. — The  reaction  of  the  freshly-voided 
normal  urine  to  litmus  paper  is  definitely  acid  from  the 
presence  of  acid  salts  and  in  particular  of  acid  sodium  phos- 
phate. An  alkaline  reaction  obtained  in  the  sample  of  a  24 
hours  specimen  which  has  been  standing  is  of  no  significance. 
An  alkaline  reaction  in  a  fresh  specimen  calls  for  further 
investigation. 

(4)  The  specific  gravity. — The  normal  specific  gravity  of 
the  urine  is  about  1,020  and  is  taken  by  means  of  the  urino- 
meter.  No  simple  examination  is  more  frequently  bungled  than 
is  this  taking  of  the  specific  gravity,  which  in  some  pathological 
states  is  of  considerable  clinical  importance.  The  following 
points  should  always  be  observed.  The  sample  examined 
must  be  one  from  the  24  hours'  output,  since  variations  in  a 
single  specimen  are  too  dependent  upon  external  circum- 
stances. It  must  be  placed  in  a  glass  sufficiently  deep 
to  contain  the  urinometer  in  almost  its  whole  length,  and 
sufficiently  wide  to  prevent  the  instrument  clinging  to  the 
sides.  The  reading  must  be  taken  with  the  eye  on  a  level 
with  the  surface  of  the  urine  and  the  surface  must  be  free 


ROUTINE   EXAMINATION— VARIATIONS.       215 

from  bubbles.  The  urinometer  itself  must  be  periodically 
inspected,  since  in  the  cheap  instrument  in  common  use  the 
little  scale  is  merely  attached  by  a  minute  piece  of  sealing- 
wax  and  may  become  considerably  dislodged  from  its  true 
position. 

(5)  The  presence  of  albumin. — Albumin  in  quantity 
detectable  by  the  ordinary  tests  is  normally  absent  from  the 
urine,  and  its  presence  in  even  small  amount  is  of  important 
clinical  significance.  The  albumin  present  in  disease  consists 
in  the  great  majority  of  cases  mainly  of  serum  albumin,  with 
varying  amounts  of  serum  globulin.  One  of  the  two  following 
tests  should  be  employed  in  every  examination,  and  either  test 
if  properly  performed  is  perfectly  reliable. 

(a)  The  heat  test. — The  urine,  if  cloudy,  must  be  filtered, 
and  if  alkaline  must  be  rendered  acid  to  litmus  with  dilute 
(33  per  cent.)  acetic  acid.  Fill  a  clean  test  tube  two-thirds 
full  with  the  clear  acid  urine.  Slowly  heat  to  boiling  point  over 
a  small  Bunsen  flame  the  top  half-inch  of  the  fluid,  rotating 
the  tube,  but  not  shaking  it.  Look  at  the  column  of  fluid 
with  the  light  coming  from  behind  and  holding  the  tube 
against  a  dark  background.  If  a  cloud,  however  faint,  appears 
in  the  heated  portion,  add  a  few  drops  of  the  acetic  acid, 
whether  the  urine  was  previously  acid  or  not.  If  the  cloud 
persists  albumin  is  present ;  if  the  turbidity  vanishes  again  it 
was  due  to  phosphates.  If  the  urine  remains  clear  after  heat-" 
ing,  add  a  little  acetic  acid;  a  slight  turbidity  due  to  albumin 
may  rarely  be  found.  If  the  urine  remains  clear,  albumin 
is  absent.  The  failure  to  examine  the  tube  against  a  dark 
background  is  responsible  for  innumerable  cases  in  which  a 
small,  but  often  important,  trace  of  albumin  has  been 
altogether  missed.  Unless  a  considerable  cloud  of  coagulable 
proteid  is  present  the  turbidity  becomes  transparent  when 
held  against  a  bright  or  comparatively  bright  ground.  Even 
should  the  urine  be  slightly  turbid  with  bacteria  (gross 
turbidity  from  this  cause  can  be  modified  by  centrifuging  at 
a  high  speed),  the  additional  cloud  of  albumin  can  be  detected 
in  this  way. 

If  the  urine  is  turbid  from  the  presence  of  phosphates  the 
turbidity  will  disappear  on  adding  the  acetic  acid.. 

If  the  turbidity  is  due  to  the  presence  of  urates  and  is  not 
completely  removed  by  filtration,  gently  warm  the  upper  half 


216  CLINICAL   PATHOLOGY. 

of  the  tube,  which  will  become  clear,  before  proceeding  to  heat 
the  top  half-inch  of  the  fluid.  If  such  a  urine  is  heated 
rapidly  the  turbidity  of  the  albumin  may  merely  replace  that 
of  the  urates  and  escape  observation. 

(6)  The  nitric  acid  test  (Heller's  test).— This  test  is 
readily  performed,  but  not  so  readily  interpreted.  It  is  further 
subject  to  certain  difficulties  not  met  with  in  the  previous  test. 
To  perform  the  reaction  place  1  inch  of  nitric  acid  in  a 
clean  test  tube.  Slowly  allow  to  run  down  the  side  of  the 
sloped  tube  about  an  equal  quantity  of  the  filtered  urine.  Allow 
to  stand  for  a  minute  or  two.  In  the  presence  of  albumin  an 
opaque  white  ring  forms  at  the  junction  of  the  two  layers  of 
fluids.  A  similar  ring  appears  if  proteoses  are  present,  but  dis- 
appears on  heating,  to  return  on  cooling.  A  diffuse  turbidity 
may  appear  if  mucin  is  present,  but  no  ring.  With  very  con- 
centrated urines  a  whitish  "  fluffy  "  opacity  with  undefined 
margins  may  form  at  the  junction  of  acid  and  urine.  In  such 
cases  the  urine  should  be  diluted  with  an  equal  volume  of 
water  and  the  test  repeated,  when  if  no  ring  forms  albumin 
is  absent.  A  coloured  transparent  ring  is  merely  due  to  the 
presence  of  urinary  pigments. 

Numerous  other  tests  are  employed  for  the  detection  of 
albumin.  Some  are  sufficiently  delicate  to  react  with  normal 
urine,  others  are  comparatively  complicated  or  require  special 
and  little  used  reagents.  It  is  advisable  to  make  use  of  one  of 
the  two  tests  given  above  and  preferably  the  former.  Which- 
ever test  is  used  care  is  essential  and  some  experience  valuable ; 
consequently  the  repeated  change  from  one  routine  test  to 
another  for  the  same  substance  is  to  be  avoided. 

(6)  The  presence  of  glucose. — The  reagents  most  com- 
monly employed  in  clinical  pathology  for  the  detection  of 
glucose  in  the  urine  are  those  of  Fehling  and  Nylander. 
The  tests  are  performed  as  follows  : — 

(a)  Fehling' S  test.— The  urine,  if  strongly  ammoniacal, 
should  first  be  rendered  acid  with  acetic  acid.  In  one  test 
tube  bring  to  the  boil  about  2  inches  of  Fehling's  solution. 
(If  the  copper  sulphate  and  soda  solutions  are  kept  in  separate 
bottles,  equal  quantities  of  each  solution  should  be  taken.) 
Boil  the  same  amount  of  urine  in  another  test  tube.  Add  the 
boiling  urine  gradually  to  the  boiling  Fehling's  solution. 
Allow  the  mixture  to  stand  till  cool  before  deciding  that  the 


ROUTINE   EXAMINATION— VARIATIONS.        217 

test  is  negative.  In  the  presence  of  glucose  a  red  or  yellow 
precipitate  of  cuprous  oxide  is  formed.  A  well-marked  precipi- 
tate occurring  on  the  addition  of  only  a  small  proportion  of 
urine  is  almost  certain  evidence  of  glucose,  but  may  be  due  to 
lactose  in  the  urine  of  nursing  women.  A  definite  precipitate 
on  cooling  after  the  addition  of  the  entire  equal  volume  of 
urine  is  probably  due  to  sugar.  A  precipitate  which  forms  only 
after  prolonged  boiling  of  the  mixture  of  urine  and  reagents, 
or  a  greenish  discoloration  and  turbidity  without  a  true 
coloured  precipitate,  may  be  due  to  traces  of  sugar  or  to  other 
substances.  The  substances  which  may  react  with  Fehling's 
solution,  and  which  are  comparatively  often  met  with  in  the 
urine,  are  uric  acid  or  creatinine  in  excess,  that  is  in  highly 
concentrated  urines,  glycuronic  acid  and  the  products  of 
certain  drugs,  of  which  the  most  important  are  chloral, 
chloroform,  carbolic  acid  and  salicylates. 
To  prepare  Fehling's  solution: — 

(1)  Powder    and   press    dry   between   filter   papers   34'64 

grammes  of  pure  copper  sulphate. 
Dissolve  in  '200  c.c.  of  warm  distilled  water.     Cool  and 
make  up  to  500  c.c. 

(2)  Dissolve    180  grammes   of  sodium   potassium  tartrate 

(Rochelle  salt)  in  300  c.c.  of  hot  water. 
Filter   and    add    70   grammes   of    pure  caustic    soda. 
Cool  and  make  up  to  500  c.c. 

(3)  Mix  equal  volumes  of  1  and  2. 

10  c.c.  of  the  mixture  is  exactly  reduced  by  '05 
gramme  of  glucose. 

(b)  Nylander's  test. — This  test  is  preferred  by  some 
because  it  is  delicate  and  is  not  affected  by  an  excess  of  uric 
acid  or  creatinine.  The  reagent  is  reduced  by  glycuronic  acid, 
lactose,  and  the  products  of  certain  drugs.  Nylander's  reagent 
is  prepared  as  follows : — 10  grammes  of  caustic  soda  are  dis- 
solved in  100  c.c.  of  water ;  4  grammes  of  sodio-potassium 
tartrate  and  2  grammes  of  bismuth  subnitrate  are  added.  After 
shaking  thoroughly,  the  mixture  is  filtered  and  kept  in  the  dark. 

To  perform  the  test  add  1  c.c.  of  the  reagent  to  10  c.c.  of 
urine.  Boil  the  mixture ;  allow  to  cool.  In  the  presence  of 
much  sugar  a  black  precipitate  of  metallic  bismuth  separates 
out ;  in  the  presence  of  small  quantities  the  urine  becomes 
dark  brown,  and  a  fine  cloud  of  bismuth  slowly  settles  down. 


218  CLINICAL   PATHOLOGY. 

In  cases  of  doubt,  and  when  it  is  particularly  desirable  to 
know  that  the  reducing  substance  is  glucose,  the  following 
scheme  of  procedure  should  be  followed. 

A  reduction  of  Fehling's  solution  having  been  obtained, 
Nylander'  test  may  be  applied,  and  if  this  is  negative  the 
reduction  of  the  Fehling's  solution  may  be  taken  as  due  to  an 
excess  of  uric  acid  or  creatinine.  If  Nylander's  solution  is  not 
readily  available  this  part  of  the  procedure  may  be  omitted. 
Next  one  portion  of  the  urine  is  preserved  and  another  portion 
is  rendered  acid  if  necessary  and  mixed  with  powdered  German 
yeast,  such  as  may  be  obtained  from  any  baker.  The  mixture 
is  placed  in  aDoremus  ureometer  tube  so  as  to  completely  fill  the 
long  limb  of  the  tube  and  about  half  the  bulb,  and  to  leave  no  air 
bubbles  at  the  top  of  the  tube.  The  tube  is  placed  in  a  warm 
place,  preferably  an  incubator  at  37°  C,  and  left  for  from  12 
to  24  hours.  A  second  tube  containing  yeast  and  water  only 
should  be  put  up  as  a  control,  since  a  small  quantity  of  gas 
may  be  given  off  from  the  yeast.  The  top  of  the  tube  is  then 
examined  for  bubbles  of  gas,  proving  that  fermentation  has 
taken  place.  The  contents  of  the  tube  are  then  filtered,  and 
Fehling's  test  is  again  performed  both  with  the  filtrate  and,  for 
the  sake  of  comparison,  with  the  reserved  and  unfermented 
portion  of  urine.  If  fermentation  has  taken  place  and  if  the 
reducing  substance  has  been  removed  by  the  process,  glucose 
was  present  in  the  urine.  Lactose,  uric  acid,  creatinine, 
glycuronic  acid,  and  the  products  of  drugs  are  not  appreciably 
affected  by  yeast. 

The  Doremus  tube  may  be  replaced  by  a  test  tube  com- 
pletely filled  and  carefully  inverted,  with  its  mouth  immersed 
in  a  beaker  of  water. 

(7)  The  nature  of  the  deposit. — If  the  sample  examined 
is  that  from  a  24  hours  specimen  a  deposit  of  some  kind  will 
probably  have  settled  down.  If,  however,  the  urine  is  perfectly 
clear,  and  no  abnormal  substances  have  so  far  been  detected 
in  it,  further  examination  is  unnecessary  as  a  routine.  If  any 
abnormal  substance,  particularly  albumin,  has  been  detected, 
or  there  is  any  reason  on  clinical  grounds  to  expect  renal 
disturbance,  a  deposit  should  be  examined  for. 

The  deposit  should  be  examined  both  by  the  naked  eye  and 
with  the  help  of  the  microscope.  The  naked-eye  examination 
of  a  deposit  is  useful,  because  it  allows  the  description  of  the 


ROUTINE   EXAMINATION— VARIATIONS.        219 

amount  of  any  abnormal  substance.  For  example,  the  centri- 
fuged  deposit  of  a  clear  urine  may  contain  leucocytes,  and  in 
the  case  of  urine  with  a  bulky  deposit  in  the  specimen  glass 
the  microscope  may  similarly  show  leucocytes.  The  difference 
is  one  of  degree  only,  but  is  a  guide  to  the  severity  of  the 
inflammatory  process.  The  diagnosis  of  a  urinary  deposit  by 
naked-eye  examination  alone  is  utterly  fallacious,  and  can 
only  be  relied  upon  by  those  who  have  never  troubled  to  prove 
the  frequency  of  error  by  exact  investigation.  It  is  true  that 
the  nature  of  a  deposit  can  be  correctly  guessed  in  a  propor- 
tion of  cases  from  the  appearance  alone,  and  that  an  abnormal 
deposit  will  often  give  an  obvious  clue  to  the  examination 
required,  but  further  investigation  by  chemical  means,  or  more 
particularly  by  the  aid  of  the  microscope,  should  never  be 
omitted. 

The  microscopical  examination  of  a  urinary  deposit  is 
readily  and  easily  made.  If  the  deposit  is  bulky  a  small 
portion  should  be  removed  from  the  bottom  of  the  specimen 
glass  by  means  of  a  pipette  and  transferred  to  the  centre  of  a 
slide.  A  cover-glass  is  carefully  let  down  on  the  drop  of 
deposit,  which  should  be  sufficient  in  amount  to  spread  across 
the  cover-slip  without  the  inclusion  of  air  bubbles,  and  not  so 
large  that  the  cover-slip  floats  about  on  the  surface  of  the  slide. 
No  staining  process  is  required.  The  slide  is  examined  with  a 
f-inch  and  with  a  ^-inch  objective.  If  the  deposit  is  slight  in 
amount  or  has  not  settled  to  the  bottom  of  the  glass  the 
supernatant  fluid  should  be  poured  off  and  the  lower  portion 
of  the  urine  shaken  up  and  then  centrifuged  at  a  moderate 
speed.  Centrifuge  tubes  should  always  be  washed  out  with 
water  before  use.  After  centrifuging  pour  off  the  supernatant 
urine  by  simply  inverting  the  tube,  and  then  shake  out  the 
last  drop  upon  the  slide.  Even  if  no  naked-eye  deposit 
appears  after  centrifuging,  the  last  drop  should  be  examined 
under  the  microscope,  since  casts,  pus  cells,  red  cells,  or  other 
bodies  may  still  be  present. 

The  above  description  applies  simply  to  the  ordinary 
routine  examination  in  all  classes  of  disease.  In  cases  where 
the  urinary  excretion  is  normal,  that  is  to  say  in  the  great 
majority  of  cases,  the  entire  examination  takes  less  than  10 
minutes.  An  account  follows  of  the  variations  from  the 
normal  which  may  be  detected  by  the  routine  examination, 


220  CLINICAL   PATHOLOGY. 

their  meaning  and  an  account  of  such  further  investigation  as 
may  be  required. 

Variations  in  the  Amount  of  Urine  passed. 

As  already  mentioned,  considerable  variations  in  the  amount 
of  urine  take  place  under  purely  physiological  conditions. 
Such  variations  are  largely  eliminated  in  the  case  of  patients 
at  rest  in  bed  on  a  fixed  diet,  and  they  will  not  be  considered 
here. 

The  urine  is  largely  increased  in  diabetes  and  very  largely  in 
the  condition  known  as  diabetes  insipidus,  a  disease  associated 
with  thirst  and  the  passage  of  large  quantities  of  pale  sugar- 
free  urine  of  a  very  low  specific  gravity.  A  considerable 
increase  in  the  urine  is  present  in  chronic  interstitial  nephritis 
with  cardiac  hypertrophy.  An  increase  is  also  commonly 
found  in  amyloid  disease,  and  may  occur  as  a  purely  functional 
condition  in  hysterical  subjects.  An  increase  may  further  be 
artificially  produced  by  diuretic  drugs. 

A  decrease  in  the  amount  of  urine  passed  occurs  in  acute 
nephritis,  when  the  output  may  be  reduced  to  a  few  ounces  or 
less  in  the  24  hours.  A  similar  but  as  a  rule  less  pronounced 
decrease  takes  place  in  cardiac  failure  with  dilatation  of  the 
right  ventricle.  Such  a  decreased  output,  due  to  cardiac  failure, 
often  occurs  in  the  last  stages  of  the  chronic  interstitial  form 
of  nephritis.  Some  decrease  in  the  urine  is  practically  always 
present  in  all  febrile  states  as  well  as  in  conditions  associated 
with  an  increased  loss  of  water  by  other  channels,  as  in 
diarrhoea  or  vomiting.  Partial  suppression  of  urine  may  also 
be  induced  by  certain  poisons,  such  as  turpentine  or  cantha- 
rides.  Decrease  in  the  urinary  output  has  to  be  distinguished 
from  retention  of  the  urine,  such  as  commonly  takes  place  in 
stricture  of  the  urethra  or  in  obstruction  due  to  an  enlarge- 
ment of  the  prostate.  Pietention  of  the  urine  may  also  occur 
in  certain  cerebral  conditions,  such  as  meningitis.  Complete 
blocking  of  both  ureters  by  calculi  leads  to  entire  suppression 
of  urine  or  "  calculous  anuria." 

Variations  in  the  Appearance. 

The  more  obvious  variations  may  be  those  of  colour  or 
those  of  transparency. 

(a)  Variations  of  colour.— The  urine  may  be  paler  than 


KOUTINE   EXAMINATION— VARIATIONS.       221 

normal,  and  specimens  of  this  kind  are  usually  those  of  a 
low  specific  gravity  associated  with  polyuria.  Diabetic  urine, 
however,  is  nearly  always  pale  and  of  a  high  specitic  gravity. 
It  has  a  somewhat  characteristic  limpid  appearance. 

Concentrated  urines  are  darker  than  normal,  and  such 
urines,  if  acid,  frequently  deposit  a  quantity  of  amorphous 
urates. 

The  most  important  urines  of  dark  colour  are  those 
associated  with  the  presence  of  blood  or  bile. 

Blood  in  the  urine  may  be  in  such  amount  as  to  colour 
the  urine  bright  red,  and  specimens  are  not  infrequently 
met  with  which  appear  to  contain  rather  more  blood  than 
urine.  Blood  in  lesser  amount  darkens  the  urine  and  gives 
it  a  peculiar  "  smoky"  "  appearance.  Blood  in  small  traces 
may  be  present  without  any  alteration  in  the  appearance  of 
the  urine. 

During  menstruation  the  examination  of  the  urine  should 
be  avoided  owing  to  its  frequent  contamination  by  blood. 
Blood  may  be  found  in  the  urine  in  numerous  conditions. 

The  blood  may  come  from  any  part  of  the  urinary  tract — 
from  a  chancre  of  the  penis,  from  the  urethra  after  injury, 
from  the  prostatic  plexus  of  veins  in  enlargement  of  the 
prostate,  from  the  bladder  in  cases  of  vesical  calculus,  tuber- 
culosis or  new  growths,  from  the  ureter  during  the  passage  of 
a  stone  or  from  the  kidneys  in  cases  of  nephritis  both  in  its 
acute  form  and  less  commonly  in  its  chronic  form,  in  eclampsia, 
in  calculus,  tuberculosis,  neoplasm,  or  in  paroxysmal 
hematuria.  Among  less  common  causes  of  hematuria  are 
hydronephrosis,  congenital  cystic  kidneys,  kinking  of  the 
kidney  over  an  abnormal  renal  artery,  injuries  to  the  kidney, 
and  acute  coli  infections  of  the  urinary  tract.  The  exact  site 
of  the  haemorrhage  can  usually  be  determined  by  an  exami- 
nation of  the  history  and  physical  signs  of  the  patient  together 
with  a  further  examination  of  the  urine,  and  in  cases  of  diffi- 
culty by  a  cystoscopic  examination.  A  further  clue  to  the 
origin  of  the  blood  may  be  obtained  by  observing  the 
relation  of  the  blood  to  the  urine  in  a  sample  as  it  is  passed. 
If  the  majority  of  the  blood  is  passed  with  the  first  portion  of 
urine  the  haemorrhage  is  probably  from  the  urethra  or 
prostate.  If  the  majority  is  passed  at  the  end  of  micturi- 
tion, it  probably  comes  from  the  bladder.     If  blood  and  urine 


222  CLINICAL   PATHOLOGY. 

are  intimately  mixed  throughout,  the  bleeding  may  be  of  renal 
origin. 

In  addition  to  the  presence  of  actual  blood  in  the  urine,  the 
same  coloration  may  be  due  to  haemoglobin  only.  The 
haemoglobin  may  be  either  oxyhaenioglobin  or  methaeruoglobin. 
Haemoglobinuria  occurs  in  black-water  fever,  and  recurrent 
paroxysmal  attacks  of  haemoglobinuria  may  take  place  in 
apparently  healthy  individuals  in  this  country,  particularly 
after  exposure  to  cold. 

The  following  tests  for  blood  and  haemoglobin  should  be 
performed  : — 

The  guiac  test. — Boil  1  inch  of  urine  in  a  test  tube. 

Allow  to  cool. 

Add  2  or  3  drops  of  tincture  of  guiacum.  A  white  pre- 
cipitate forms. 

Pour  gently  down  the  side  of  the  tube  1  inch  of  ozonic 
ether. 

Allow  to  stand. 

A  blue  ring  appears  at  the  junction  of  urine  and  ether  if 
blood  is  present. 

This  test  is  a  fairly  delicate  one  for  the  presence  of  blood  or 
haemoglobin.  It  is  given  also  by  the  urine  of  a  patient  who 
is  taking  iodides.  In  the  presence  of  pus  in  any  quantity  a 
greenish  blue  ring  is  given.  Since  the  test  is  not  infallible, 
and  may  be  given  by  either  blood  or  haemoglobin,  and  may 
not  be  given  in  the  presence  of  minute  quantities  of  blood,  one 
or  both  of  the  following  tests  should  always  be  performed  in 
addition. 

The  microscopic  test. — Centrifuge  the  urine  if  necessary. 

Place  a  drop  of  the  deposit  on  a  slide  and  cover  with  a 
cover-glass.  Examine  under  a  ^-inch  objective  with  the 
diaphragm  partly  closed.  Eed  blood  corpuscles  should  be 
recognised  with  certainty,  even  if  very  few  are  present.  Should 
the  student  be  in  any  doubt  as  to  whether  the  corpuscles  seen 
are  red  cells  or  not  he  should  make  a  dried  film  preparation 
and  stain  with  Leishman's  stain.  The  red  cells  in  such  a 
preparation  are  commonly  distorted,  but  retain  their 
characteristic  staining  reaction. 

The  microscopic  test  is  the  only  infallible  one  for  the 
presence  of  blood,  and  is  very  easily  performed.  In  cases  of 
pure  hemoglobinuria  no  red  cells  may  be  found,  but  the  guiac 


KOUTINE   EXAMINATION— VARIATIONS.        223 

test  will  be  positive.  An  occasional  red  cell  may  be  found  in 
a  centrifuged  deposit  when  the  guiac  test  is  negative. 

The  spectroscopic  test.— With  a  positive  guiac  reaction 
and  a  proportional  number  of  red  cells  in  the  urinary  deposit 
it  is  unnecessary  to  confirm  the  result  by  the  spectroscope.  In 
cases  of  supposed  hemoglobinuria  the  spectrum  of  the  urine 
must  always  be  examined.  A  small  direct-vision  spectroscope 
can  be  used,  and  the  urine  if  very  deeply  tinged  should  be 
diluted  with  water.  If  the  substance  present  is  methemoglobin 
a  band  is  seen  in  the  red  between  C  and  D  in  addition  to  the 
two  bands  of  oxyhemoglobin  (see  page  93). 

Bile  in  the  urine  is  another  common  cause  of  alteration  of 
colour.  Bile  is  present  in  the  urine  in  every  variety  of 
jaundice  with  the  exception  of  congenital  family  cholemia. 
Bile  salts  are  usually  but  not  always  present  as  well  as  the 
pigment.  Bile  in  the  urine  is,  as  a  rule,  fairly  obvious  to  the 
naked  eye,  and  if  a  test  tube  containing  the  urine  is  shaken  a 
greenish-yellow  froth  appears  at  the  top. 

The  following  tests  for  bile  should  be  performed : — 

Gmelin's  test. — Filter  several  cubic  centimetres  of  urine 
through  a  clean  filter  paper.  When  all  the  urine  has  passed 
through  dip  a  glass  rod  in  yellow  nitric  acid  and  then  on  the 
filter  paper. 

A  spreading  ring  of  colours  appears  round  the  area  touched 
by  the  acid.  The  colours  are  from  within  outwards  yellow, 
red,  violet  and  green.  The  test  is  not  positive  unless  the 
green  colour  is  certainly  seen. 

Should  the  bile  be  present  in  small  amount  a  positiva 
reaction  may  be  obtained  if  a  considerable  bulk  of  urine  is 
filtered  two  or  three  times  through  the  paper. 

Iodine  test. — Take  2  c.c.  of  urine  in  a  test  tube. 

Carefully  pour  on  the  top  of  the  urine  2  c.c.  of  tincture  of 
iodine. 

A  green  ring  appears  at  the  junction  of  the  liquids. 

Both  the  above  tests  are  for  the  presence  of  bile  pigments. 
A  simple  and  fairly  delicate  test  for  the  presence  of  bile  acids 
is  the  following  : — 

Hay's  test. — Pour  a  few  cubic  centimetres  of  urine  into  a 
watch  glass. 

Gently  sprinkle  on  to  the  surface  of  the  urine  a  little  flowers 
of  sulphur. 


224  CLINICAL  PATHOLOGY. 

In  the  case  of  normal  urine  the  sulphur  floats.  "When  bile 
salts  are  present  the  surface  tension  of  the  urine  is  lowered 
and  the  sulphur  sinks. 

For  clinical  purposes  Hay's  test  is  sufficiently  accurate. 

Pettenkofer's  test,  using  cane  sugar  and  sulphuric  acid  as 
the  reagents,  is  not  very  satisfactory  with  urine  as  the  test 
fluid. 

Urobilin  may  cause  a  darkening  of  the  colour  of  the  urine. 
It  is  not  present  in  any  quantity  in  normal  freshly-voided 
urine.  It  may  be  present  in  excess  with  fever,  in  pernicious 
anaemia,  in  congenital  family  cholaernia,  and  in  other  diseases. 
If  urobilin  is  present  in  considerable  excess  the  absorption 
band  of  the  spectrum,  which  appears  at  the  junction  of  the 
green  and  blue,  can  be  seen  in  direct  examination  of  the  fresh 
urine  with  the  direct-vision  spectroscope.  The  normal  urine 
shows  no  bands  other  than  a  general  darkening  of  the  violet 
end  of  the  spectrum.  Urobilin  present  in  lesser  amount  can 
be  demonstrated  as  follows  : — 

Saturate  the  urine  with  ammonium  chloride  to  precipitate 
the  urates. 

Filter. 

Saturate  the  filtrate  with  ammonium  sulphate. 

Add  a  drop  of  sulphuric  acid. 

Shake  with  a  mixture  of  2  parts  ether  and  1  part 
chloroform. 

Pipette  off  the  ether-chloroform  layer  and  examine  for  the 
urobilin  spectrum. 

Among  other  and  rarer  causes  of  dark  urines  are  alkapton, 
hsematoporphyrin,  melanin  and  carbolic  acid. 

Alkaptonuria  is  a  rare  congenital  abnormality  which 
persists  for  life  and  is  attended  by  no  symptoms.  The  urine 
is  of  normal  colour  when  passed  and  becomes  of  a  dark,  almost 
black,  colour  on  standing.  The  urine  readily  reduces  Fehling's 
solution  and  darkens  Nylander's  solution,  but  without  the 
production  of  a  precipitate.  The  urine  is  not  fermented  by 
yeast.  Ferric  chloride  added  to  the  urine  drop  by  drop 
produces  a  striking  and  momentary  deep  blue  colour. 

Haematoporphyrinuria. — Haematoporphyrin  is  present  in 
the  normal  urine,  but  only  in  very  small  amount.  In  the  condi- 
tion known  as  haematoporphyrinuria  the  urine  is  obviously  dark 
and  may  be  of  a  deep  port  wine  colour.    The  condition  arises  in 


EOUTINE    EXAMINATION— VAEIATIONS.        225 

patients  who  have  been  taking  sulphonal  in  excess,  and  from 
this  cause  has  occurred  in  almost  epidemic  form  in  lunatic 
asylums.  Unless  the  condition  is  recognised  at  once  and  the 
sulphonal  stopped  death  is  likely  to  result.  A  urine  contain- 
ing haematoporphyrin  in  excess  does  not  reduce  Fehling's 
solution  and  does  not  give  the  guiac  reaction.  If  the  pigment 
is  present  in  great  excess  direct  examination  of  the  urine  shows 
the  spectrum  of  alkaline  haBmatoporphyrin.  The  spectrum 
has  4  bands — one  between  C  and  D,  one  at  D,  one  just  on  the 
red  side  of  E,  and  a  broad  band  between  the  green  and  the  blue. 
If  the  spectrum  is  not  clearly  obtained,  extract  the  pigment  in 
the  following  manner  : — 

To  100  c.c.  of  urine  add  20  c.c.  of  dilute  soda. 

Allow  the  precipitate  of  pigment  and  earthy  phosphates 
to  settle. 

Decant  the  supernatant  fluid. 

Transfer  precipitate  to  a  filter  paper  and  wash  with  water. 

Extract  precipitate  with  alcohol  acidified  with  hydrochloric 
acid. 

Examine  extract  for  spectrum  of  acid  haBmatoporphyrin. 

The  spectrum  of  acid  haBmatoporphyrin  has  2  bands — a 
narrow  band  in  the  orange  near  the  D  line,  and  a  broad  band 
in  the  yellow. 

Melaninuria  is  a  rare  condition  which  may  be  found  in 
patients  with  melanotic  sarcomata,  and  occasionally  with  non- 
malignant  pigmented  papillomata  of  the  skin.  The  urine  as 
a  rule  is  dark  when  passed,  but  becomes  considerably  darker 
on  exposure  to  the  air.  The  addition  of  a  few  drops  of  nitric 
acid  causes  immediate  blackening  of  the  urine.  No  reduction 
of  Fehling's  solution  is  produced  and  no  reaction  with  the 
guiac  test.  There  is  no  spectrum.  A  dark  urine  which  fulfils 
these  conditions  in  all  probability  contains  melanin,  but  there 
is  no  simple  and  satisfactory  direct  test  for  this  substance. 

Carboluria  may  occur  in  any  variety  of  carbolic  acid 
poisoning.  With  susceptible  patients  the  absorption  of  a 
very  small  quantity  of  carbolic  acid,  such  as  takes  place  after 
the  application  of  a  "  carbolic  cap  "  to  the  head,  may  lead  to 
the  production  of  carboluria.  The  patient  may  show  little  or 
no  symptoms.  The  urine  becomes  considerably  darker  on 
standing  or  after  the  addition  of  nitric  acid,  and  in  well- 
marked  cases  is  of  a  distinctive  greenish-blue  colour.     This 

p.  15 


226  CLINICAL  PATHOLOGY. 

colour  and  the  evidence  of  exposure  to  carbolic  acid  helps  to 
distinguish  the  urine  from  that  of  melaninuria.  The  pigments 
produced  in  the  urine  by  carbolic  poisoning  are  chemically 
similar  to  those  of  alkaptonuria.  Fehling's  solution  may  be 
reduced. 

Certain  urines  of  striking  appearance  sometimes  met  with 
follow  the  absorption  of  harmless  or  comparatively  harmless 
substances. 

Methylene  blue  taken  by  the  mouth,  or  given  hypo- 
dermically,  leads  to  the  production  of  a  remarkable  bright 
green  (not  blue)  urine.  There  is  at  the  present  a  considerable 
epidemic  of  these  urines  owing  to  the  activity  of  the  quack 
medicine-monger  who  sells  methylene  blue  "  kidney  pills  "  to 
purge  the  urine  of  the  credulous.  The  coloration  of  the 
urine  may  persist  for  several  days  after  taking  the  drug  and 
is  quite  harmless.  The  mental  shock  to  a  neurotic  patient  on 
passing  bright  green  water  may,  however,  be  considerable. 

Eosin  has  been  extensively  used  as  a  coloring  matter  for 
cheap  sweets,  and  a  curiously  dichroic  urine  may  result.  The 
tint  of  the  urine  is  exactly  that  of  a  dilute  solution  of  eosin 
and  is  easily  recognised. 

Among  other  substances  santonin  may  produce  a  greenish 
urine,  and  rhubarb  or  senna  a  reddish-brown  coloration. 
Resorcin  used  as  an  ointment  may  lead  to  a  striking  greenish 
coloration  of  the  urine. 

(b)  Variations  of  transparency. — Among  the  causes  of 
turbidity  of  the  urine  are  the  following. 

Urates  are  a  frequent  cause  of  turbidity  of  the  urine, 
particularly  on  standing,  the  urates  separating  out  as  the 
urine  cools.  The  separation  of  urates  is  the  rule  in  acid, 
concentrated  urines,  whatever  may  be  the  cause  of  the  con- 
centration. Turbidity  due  to  urates  is  readily  recognised, 
since  on  warming  the  urine  it  becomes  clear. 

Phosphates  may  produce  a  turbidity  in  neutral  or  alkaline 
urines.  On  acidifying  the  urine  with  dilute  acetic  acid  the 
turbidity  goes. 

Bacteria  commonly  produce  a  turbidity  in  urines  which 
have  been  standing  exposed  to  the  air ;  their  presence  in  such 
urines  is  of  no  significance.  In  pathological  conditions  the 
urine  may  be  turbid  from  the  presence  of  bacteria  at  the  time 
of  passage      The  turbidity  has  the  exact  appearance  of  that 


ROUTINE   EXAMINATION— VARIATIONS.        227 

produced  in  a  broth  culture  tube  by  the  growth  of  organisms. 
It  does  not  alter  on  warming  or  on  the  addition  of  an  acid,  nor 
is  it  removed  by  nitration.  It  is  little  affected  by  centrifuging, 
except  at  a  very  high  speed.  A  drop  of  the  urine,  or  pre- 
ferably of  the  centrifuged  deposit,  examined  under  the 
microscope,  is  seen  to  be  swarming  with  bacilli  or  cocci. 
The  examination  of  urines  for  bacteria  will  be  described 
subsequently. 

Fat  in  the  urine  or  lipuria  is  an  extremely  rare  condition. 
The  fat  may  be  present  in  such  amount  that  the  urine  looks 
like  milk,  and  on  standing  a  creamy  layer  rises  to  the  top. 
Lipuria  of  this  degree  is  usually  due  to  the  presence  of  filarise, 
the  embryos  of  which  should  be  looked  for  in  the  urine  and 
in  the  blood.  In  this  variety  of  lipuria,  which  is  commonly 
known  as  chyluria,  because  the  fat  escapes  from  a  ruptured 
lymph  vessel,  the  fat  may  appear  as  amorphous  granules 
under  the  microscope.  Lipuria,  in  appreciable  degree,  may 
very  rarely  accompany  diabetes,  pregnancy,  and  phosphorus 
poisoning.  It  has  been  described  in  other  conditions.  The 
fat  in  such  cases  may  be  in  the  form  of  oil  droplets.  (A  few 
oil  drops  in  the  urine  are  almost  the  rule  in  catheter 
specimens  and  come  from  the  lubricating  fluid  used  for  the 
catheter.)  In  the  case  of  milky  urines  it  is  always  advisable 
to  make  sure  that  no  fat-containing  substance,  such  as  milk, 
has  been  added  to  the  urine  by  the  patient.  In  cases  of 
doubt  a  catheter  must  be  passed. 

Turbidity  of  the  urine  due  to  fat  can  be  removed  by 
extraction  with  ether,  and  the  presence  of  fat  in  the  ether 
extract  should  be  verified. 


15—2 


CHAPTEE    XVII. 

variations  in  acidity,  and  acidosis variations   in   specific 

gravity — urea — proteids carbohydrates. 

Variations  in  Acidity. 

Variations  in  the  ammonia  nitrogen  (acidosis). — 

The  normal  acid  reaction  to  litmus  paper  may  be  converted 
into  an  alkaline  one  by  the  growth  of  staphylococci  in  the 
urine  on  standing.  The  change  in  reaction  results  from  the 
conversion  of  urea  into  ammonium  carbonate.  The  normal 
urine  also  may  be  faintly  alkaline  at  the  height  of  digestion, 
and  may  be  rendered  alkaline  by  certain  drugs  such  as 
potassium  citrate.  The  habitual  passage  of  an  alkaline  urine 
is  abnormal,  and  in  the  majority  of  cases  is  due  to  the  growth 
of  organisms  in  the  urinary  tract.  The  organisms  found  in 
an  alkaline  urine  are  usually  staphylococci,  or  the  bacillus 
proteus,  and  the  site  of  infection  is  more  commonly  the  bladder 
than  the  kidney.  Infections  with  the  bacillus  coli  are  almost 
always  accompanied  by  an  acid  urine. 

In  order  to  measure  the  acidity  of  the  urine  phenol-phthalein 
may  be  used  as  an  indicator,  and  sufficient  deci-normal  soda 
run  into  a  measured  quantity  of  urine  until  neutralisation  is 
effected.  The  estimation  of  the  acidity  is  the  first  stage  in 
the  estimation  of  the  ammonia  nitrogen  by  an  accurate  and 
simple  method.     The  procedure  is  as  follows  : — 

Measure  out  25  c.c.  of  urine  into  a  beaker  and  dilute  with 
about  double  that  volume  of  distilled  water. 

Add  2  or  3  drops  of  phenol-phthalein. 

N 
Run  in  r-pr  NaOH  from  a  burette  until  a  faint   permanent 

pink  colour  is  produced. 

Note  the  number  of  cubic  centimetres  of  NaOH  used. 

Measure  about  10  c.c.  of  commercial  (40  per  cent.)  formalin 
into  a  second  beaker. 

Add  phenol-phthalein. 

N 
Neutralise  exactly  with  —  NaOH. 


VAKIATIONS   IN   ACIDITY  AND   ACIDOSIS,    ETC.    229 

Add  the  neutral  formalin  to  the  neutral  urine. 

The  pink  colour  disappears. 

.     N 
Run  m  r^r  NaOH  until  the  pink  colour  returns. 

Note  the  number  of  cubic  centimetres  of  NaOH  used. 

N 
The  result  is  calculated  in  terms  of  j^.  NaOH  for  the  acidity. 

That  is  to  say,  the  acidity  of  the  urine  is  given  as  the  number  of 

N 
cubic  centimetres  of  r-x  NaOH  required  to  neutralise  100  c.c. 

of  urine  to  phenol-phthalein.  Thus  if  10  c.c.  of  soda  were  used 
in  the  first  titration  to  neutralise  25  c.c.  of  urine,  the  acidity 

of  the  urine  is  —  X  100  =  40.     The  ammonia  result  should 

be  expressed  in  grammes  of  ammonia  per  24  hours.  The 
number  of  cubic  centimetres  of  soda  used  in  the  second 
titration  of  the  urine  is  the  equivalent  of  the  number 
of  cubic  centimetres  of  ammonia  present  in  the  25  c.c.  of 
urine.  Supposing  the  number  of  cubic  centimetres  of  soda 
used  in  the  second  titration  to  have  been  10,  then    10  c.c. 

^NaOH  =  10     c.c.  ^  NH8  =  10  X  -0017      gramme      NH3. 

Therefore  the  ammonia  passed  in  the  24  hours  =  10  X  #0017 

24  hours'  urine  in  cubic  centimetres 
X  "  ~^5~~ 

The  reaction  depends  upon  the  combination  of  the  ammonium 
salts  with  formaldehyde  to  form  urotropin,  and  the  consequent 
liberation  of  the  acids  previously  combined  with  ammonia.  The 
method  is  an  extremely  accurate  one,  but  yields  results  higher 
than  those  obtained  by  the  older  and  more  complicated  method 
of  Folin,  since  the  amino  groups  of  the  amino-acids  react  with 
the  formaldehyde  and  are  included  in  the  ammonia  total. 

Variations  in  the  acidity  and  ammonia  nitrogen  of  the  urine 
are  of  considerable  clinical  significance,  and  may  be  very 
marked  in  any  of  the  conditions  which  may  be  associated  with 
"  acidosis."  The  most  important  pathological  states  of  which 
acidosis  may  be  a  feature  are  starvation,  eclampsia,  diabetes, 
and  chloroform  poisoning.  A  relative  increase  in  the  ammonia 
nitrogen  is  the  rule  in  eclampsia  and  in  the  condition  known 
as  delayed  chloroform  poisoning.  The  onset  of  coma  in 
diabetes    is   similarly   accompanied    by   the    appearance    of 


230  CLINICAL   PATHOLOGY. 

abnormal  acids  in  the  urine.  The  more  important  acids 
which  may  be  found  in  the  urine  in  such  cases  are  diacetic 
and  B-oxybutyric  acids.  The  excess  of  ammonia  nitrogen  in 
the  urine  is  commonly  explained  as  following  the  excessive 
production  of  ammonia  needed  to  combine  with  the  abnormal 
acids. 

The  presence  of  acidosis  can  be  proved  by  an  examination 
of  the  urine,  and  its  onset  can  frequently  be  detected  before 
the  clinical  state  is  manifest. 

The  acidity  of  the  urine  as  estimated  by  a  simple  titration 
with  soda  and  phenol-phthalein  is  too  variable  to  be  of  much 
clinical  assistance. 

The  estimation  of  the  ammonia  output  alone  has  much  less 
significance  than  a  comparison  between  the  percentage  of 
ammonia  nitrogen  and  the  percentage  of  the  total  nitrogen  in 
the  urine.  Under  normal  conditions  the  ammonia  nitrogen 
forms  about  3  per  cent,  of  the  total  nitrogen,  and  in  acidosis 
considerably  more.  In  order  to  estimate  the  total  nitrogen  it 
is  necessary  to  use  Kjeldahl's  method.  Kjeldahl's  method  is 
not  given  here  because  it  is  too  difficult  for  ordinary  clinical 
use  in  that  it  requires  a  somewhat  cumbrous  apparatus  with 
which  the  operator  must  be  thoroughly  conversant.  A  reliable 
guide  to  the  extent  of  the  acidosis  can  be  obtained  from  a 
comparison  of  the  ammonia  and  the  urea  nitrogen.  The  urea 
is  readily  estimated  by  one  of  the  methods  to  be  described 
shortly.     The  calculation  is  made  as  follows  : — 

The   molecular  weight  of  ammonia  or  NH3  being  17,  the 

nitrogen  fraction  of  ammonia  is  ^. 

The  molecular  weight  of  urea  or  CO/,.2  being  60,  the 

28        7 
nitrogen  fraction  is  -ttc  or  ^. 

The  amount  of  ammonia  in  grammes  in  a  given  sample  is 
estimated  by  the  formalin  method,  and  fourteen-seventeenths 
of  this  represents  the  ammonia  nitrogen.  The  urea  is  similarly 
calculated  in  grammes  in  the  same  sample  of  urine,  and  seven- 
fifteenths  of  this  weight  is  nitrogen. 

Under  normal  conditions  the  ammonia  nitrogen  is  about 
one-twentieth  of  the  urea  nitrogen,  and  in  conditions  asso- 
ciated with  acidosis  it  may  rise  to  one-fourth.     A  rise  in  the 


VARIATIONS  IN   ACIDITY  AND   ACIDOSIS,   ETC.    231 

ammonia  nitrogen  to  one-tenth  of  the  urea  nitrogen  is  definite 
evidence  of  acidosis. 


Vaeiations  in  Specific  Geavity. 

Variations  in  the  density  of  the  urine  observed  in  catheter 
specimens  or  single  samples  are  of  little  significance.  The 
specific  gravity  must  be  taken  from  the  sample  of  a  24  hours 
specimen  of  urine. 

The  specific  gravity  of  the  urine  is  unaffected  by  substances 
in  suspension,  and  very  little  affected  by  any  albumin  that 
may  be  present.  It  is  dependent  upon  the  salts  in  solution, 
and  varies  directly  with  the  amount  of  urea  present. 

The  specific  gravity  is  raised  in  practically  all  conditions 
associated  with  a  diminished  urinary  output,  such  as  occurs 
during  violent  exercise,  with  pyrexia,  in  acute  nephritis  and  in 
cardiac  failure.  An  increased  urinary  output  with  a  urine  of 
high  specific  gravity  is  characteristic  of  diabetes.  In  this  disease 
the  specific  gravity  is  commonly  from  1,035  to  1,045,  and  may 
rise  higher. 

The  specific  gravity  is  generally  lowered  when  the  urinary 
output  is  increased,  as  in  chronic  interstitial  nephritis  and 
amyloid  disease.  The  passage  of  a  large  quantity  of  urine  of 
a  specific  gravity  constantly  at  or  below  1,010,  and  containing 
a  trace  of  albumin,  is  characteristic  of  chronic  interstitial 
nephritis. 

Variations  in  the  specific  gravity  must  always  be  considered 
in  conjunction  with  the  amount  of  urine  passed  and  the 
percentage  of  urea  present,  and  repeated  observations  must 
be  made  before  deductions  of  any  value  can  be  drawn. 

The  Urea. 

The  amount  of  urea  excreted  by  the  kidneys  varies  within 
fairly  wide  limits,  and  the  variations  naturally  depend  to  a 
large  extent  upon  the  amount  of  nitrogen  in  the  food  and 
upon  the  activity  of  the  individual.  A  patient  at  rest  in  bed 
on  a  milk  diet  will  excrete  less  urea  than  a  similar  patient  at 
work  on  an  ordinary  mixed  diet.  The  majority  of  urea 
estimations  performed  on  the  urine  of  patients  in  hospital 
wards  in  the  usual  perfunctory  manner  are  scarcely  worth  the 


232  CLINICAL   PATHOLOGY. 

sodium  hypobrornite  expended  on  them,  yet  if  due  allowance 
be  made  for  the  condition  of  the  patient  valuable  clinical 
information  can  be  obtained  from  urea  estimations  in  suitable 
cases. 

The  exact  estimation  of  the  urea  in  a  sample  of  urine  is  a 
matter  of  some  difficulty,  but  the  results  obtained  by  one  of 
the  following  methods  are  sufficiently  accurate  for  all  clinical 
purposes.  Under  normal  conditions  the  urine  contains  about 
2  per  cent,  of  urea,  and  the  daily  excretion  is  from  30  to  40 
grammes. 

Estimations  of  urea  should  always  be  made  if  possible  from 
a  measured  24  hours  specimen  of  urine.  The  deductions  to  be 
drawn  from  these  estimations  will  be  considered  later.  The 
methods  described  depend  upon  the  decomposition  of  urea  by 
alkaline  hypobroniite  into  nitrogen  and  carbon  dioxide.  The 
carbon  dioxide  is  absorbed  by  the  alkali,  and  the  nitrogen  is 
collected  and  measured.  The  whole  of  the  nitrogen,  however, 
is  not  evolved  by  this  method,  and  whereas  1  gramme  of  urea 
should  yield  373  c.c.  of  nitrogen,  only  354  c.c.  are  actually 
evolved,  or  about  92  per  cent.  When  sugar  is  present  in  the 
urine  a  greater  yield  of  the  nitrogen,  or  about  99  per  cent.,  is 
obtained.  The  scale  of  the  ureometers  in  common  use  is 
corrected  for  the  yield  of  normal  urine,  consequently  in  cases 
of  diabetes  the  results  are  too  high,  and  should  be  multiplied 
,  92 
by99' 

Before  making  the  estimation  the  urine  should  be  tested  for 
albumin,  and  if  more  than  a  trace  is  present  a  measured 
quantity  of  urine  is  taken,  acidified  with  acetic  acid,  raised  to 
the  boiling  point,  cooled,  filtered,  and  made  up  to  the  original 
volume  with  distilled  water.  If  much  albumin  is  present  the 
urine  must  be  diluted  before  boiling. 

The  estimation  should  be  carried  out  by  one  of  the  two 
forms  of  apparatus  described  below  : — 

(1)  Gerrard's  ureometer. — The  apparatus  consists  of  a 
graduated  glass  cylinder,  the  open  top  of  which  is  fitted  with  a 
rubber  cork,  through  which  passes  a  glass  T-piece.  One  limb 
of  the  T-piece  has  a  short  piece  of  rubber  tubing  attached, 
which  can  be  closed  by  a  clip.  The  other  limb  is  connected 
with  a  long  rubber  tube,  which  passes  by  means  of  a  glass 
junction   through    a   rubber   cork,    filling    the    mouth    of    a 


VARIATIONS   IN   ACIDITY   AND   ACIDOSIS,   ETC.     233 


glass  bottle  (a).     The  glass  bottle  contains  a  small  tube  marked 

to  contain  5  c.c.     At  the  lower  end  of  the  glass  cylinder  is  an 

opening  which  connects  by  a  piece  of  rubber  tubing  to  the 

bottom  of  a  wide  glass 

tube  (c).      The    glass 

tube     is    attached    to 

the  cylinder  by  a  metal 

fitting,    and     can     be 

pushed  up  and  down 

upon  the  cylinder. 

The  hypobromite 
solution  used  must  be 
freshly  made,  other- 
wise it  will  decom- 
pose and  give  off  gas. 
The  bromine  and  the 
caustic  soda  should 
therefore  be  kept 
separate  and  mixed  as 
required.  Bromine  is 
supplied  in  glass  cap- 
sules holding  2'5  c.c. 
To  mix,  place  25  c.c. 
of  40  per  cent,  caustic 
soda  in  a  strong  glass 
bottle  fitted  with  a 
glass  stopper.  Place 
one  of  the  bromine 
capsules  in  the  bottle. 
Fit  the  stopper  tightly. 
Shake  the  bottle 
smartly  so  as  to  crack 
the  bromine  capsule.  Do  not  use  weak  caustic  soda,  otherwise 
the  irritating  bromine  vapour  will  escape. 

To  make  the  estimation : — 

Place  the  25  c.c.  of  hypobromite  solution  in  the  glass  bottle. 

Measure  5  c.c.  of  the  urine  into  the  small  tube. 

Wipe  the  outside  of  the  tube  and  stand  it  carefully  in  the 
bottle  so  that  the  urine  does  not  mix  with  the  hypobromite. 

Do  not  cork  the  bottle. 

Pour  water  into  the  wide  tube. 


Fig.  16. — Gerrard's  Ureometer. 


284 


CLINICAL   PATHOLOGY. 


Now  cork  the  bottle  tightly. 

Open  the  clip  on  the  T-piece. 

Eaise  or  depress  the  wide  tube  until  the  water  in  the 
graduated  cylinder  is  at  the  zero  mark,  and  level  with  the 
water  in  the  tube. 

Close  the  clip  on  the  T-piece. 

Tilt  the  bottle  so  that  the  urine  mixes  with  the  hypobromite. 


Fig.  17. — Mayhew's  Ureoraeter. 


Wait  for  15  minutes,  giving  the  bottle  an  occasional  shake 
after  the  first  effervescence  has  subsided. 

Lower  the  tube  until  the  water  in  tube  and  cylinder  are 
again  level. 

Bead  off  the  amount  of  nitrogen  in  the  cylinder. 

The  scale  is  graduated  in  percentages  of  urea. 

The  apparatus  must  be  tested  from  time  to  time  to  make 
sure  that  there  is  no  leakage  in  any  of  the  rubber  connections 
or  their  attachments. 

The  same  hypobromite  may  be  used  for  a  second  estimation 
if  required. 

(2)  Mayhew's  ureometer. — This  simple  form  of  apparatus 


VABIATIONS   IN   ACIDITY   AND  ACIDOSIS,   ETC.     235 

is  very  convenient  for  clinical  purposes.  The  apparatus  con- 
sists of  a  glass  cylinder  composed  of  one  long  straight  central 
part,  which  is  graduated,  and  two  shorter  terminal  parts  bent 
at  an  angle  to  the  graduated  part.  The  short  limb  nearest  to 
the  zero  mark  has  a  wide  mouth  provided  with  a  well-fitting 
rubber  cork.  A  small  glass  tube  graduated  to  contain  1  c.c. 
of  urine  completes  the  apparatus. 

To  make  the  estimation  : — 

Prepare  hypobromite  solution  as  for  Gerrard's  apparatus. 

Pour  hypobromite  into  the  wide-mouthed  opening  in  such 
amount  that  when  the  long  limb  is  horizontal  the  fluid  rises 
about  halfway  in  each  of  the  short  limbs. 

Fit  in  the  cork  and  hold  the  long  limb  vertical  with  the 
stoppered  end  uppermost.  The  hypobromite  level  should  be 
at  the  zero  mark  in  the  long  limb,  and  there  should  be  several 
cubic  centimetres  in  the  upper  shorter  limb,  with  a  consider- 
able space  between  the  two  volumes  of  fluid. 

Place  the  apparatus  against  a  rest  with  the  long  limb 
horizontal  and  remove  the  cork. 

Fill  the  small  glass  tube  with  urine  to  the  1  c.c.  mark. 

Wipe  the  outside  of  the  tube. 

Holding  the  apparatus  horizontal  in  the  left  hand,  slip  the 
glass  tube  into  the  wide-mouthed  limb.  The  foot  of  the  tube 
should  be  in  the  hypobromite,  but  the  mouth  of  the  tube  above 
the  level  of  the  solution,  so  that  urine  and  solution  do  not 
mix. 

Fit  in  the  rubber  cork. 

Alter  the  long  limb  in  one  movement  from  the  horizontal  to 
the  vertical  position. 

Hang  up  the  apparatus  and  leave  for  15  minutes. 

The  urine  mixes  with  the  hypobromite.  The  nitrogen  is 
liberated  into  the  upper  bend  of  the  apparatus  and  forces  the 
solution  down  the  graduated  limb. 

Head  the  level  of  the  fluid  in  the  graduated  limb.  The  scale 
is  graduated  in  percentages  of  urea  as  well  as  (in  most  forms 
of  the  apparatus)  in  grains  per  ounce. 

The  calculation  should  always  be  worked  out  for  the  urea 
output  of  the  24  hours.  The  statement  that  a  certain  sample 
of  urine  contains  a  certain  percentage  of  urea  conveys  very 
little  information.  The  variations  in  urea  content  of  different 
samples  of  urine  from  the  same  patient  over  a  period  of  24 


236  CLINICAL   PATHOLOGY. 

hours  are  considerable.  Consequently  a  mixed  sample  of  the 
urine  must  be  taken,  and  the  amount  of  the  urine  must  be 
measured.  Also  the  diet  of  the  patient  must  so  far  as  practic- 
able be  taken  into  consideration. 

Among  the  conditions  in  which  the  urea  output  is  increased 
are  fevers  and  diabetes. 

More  important  are  the  conditions  in  which  the  urea  output 
is  diminished.  These  include  both  medical  and  surgical  affec- 
tions of  the  kidney.  The  factors  of  the  diet  and  the  general 
circumstances  of  the  patient  having  been  taken  into  account, 
the  urea  output  may  be  taken  as  a  rough  but  useful  clinical 
guide  to  the  activity  of  the  kidneys.  Slight  variations  in  the 
amount  of  urea  are  of  little  moment,  and  in  all  cases  a  series 
of  observations  should  be  made  without  alteration  of  diet.  In 
both  acute  and  chronic  nephritis  the  urea  output  is  diminished. 
In  acute  nephritis  the  diminution  is  a  well-marked  feature, 
and  a  rise  in  the  urea  excretion  is  evidence  of  improvement. 
In  surgical  affections  of  the  urinary  tract  urea  estimations 
are  frequently  of  value.  In  cases  of  prostatic  enlarge- 
ments with  back  pressure  some  estimation  should  always  be 
made  of  the  amount  of  normal  renal  tissue  available  to  the 
patient.  The  general  clinical  condition  gives  a  fair  idea  of 
the  state  of  the  kidneys,  but  the  clinical  estimate  should 
always  be  aided  by  examination  of  the  urine.  It  may  be 
stated  roughly  that  it  is  dangerous  to  operate  upon  such 
patients  when  the  specific  gravity  is  constantly  less  than 
1,008  and  the  urea  less  than  0*6  per  cent,  of  an  average 
measurement.  When  the  specific  gravity  is  above  1,010  and 
the  urea  percentage  about  l'O  or  over,  operation  should  be 
successful.  It  is  occasionally  necessary  to  estimate  the  urea 
in  a  ureteric  catheter  specimen,  and  particularly  when  informa- 
tion is  required  as  to  which  kidney  is  diseased,  or  as  to 
whether  the  diseased  kidney  is  functionating.  The  urea 
percentages  in  such  specimens  should  be  interpreted  with 
caution,  since  slight  differences  on  the  two  sides  are  found 
in  health,  but  gross  differences  may  be  of  great  assistance  in 
diagnosis. 

Attempts  have  been  made  to  provide  a  more  exact  clinical 
method  by  which  the  excretory  activity  of  the  kidneys  can  be 
estimated.  A  function  of  the  kidneys  is  to  remove  waste 
products   from    the    body,  and   if  they    fail  to    do  so   these 


VARIATIONS   IN   ACIDITY   AND   ACIDOSIS,   ETC.     237 

products  accumulate  in  the  blood.  The  most  reliable  method 
therefore  of  investigation  involves  a  comparison  of  the  blood 
and  the  urine.  Under  normal  conditions  the  salts  in  solution 
in  the  urine  are  double  those  in  solution  in  the  blood.  Under 
abnormal  conditions  the  ratio  may  be  actually  reversed.  The 
ratio  between  the  inorganic  salts  in  solution  in  the  urine  and 
those  in  the  blood  is  known  as  the  haemorenal  index. 
The  estimation  of  the  salts  in  the  blood  and  urine  can  be 
performed  in  one  of  two  ways.  A  direct  analysis  is  not  practi- 
cable, since  the  amount  of  blood  available  is  very  small.  One 
method  consists  in  taking  the  freezing  points  of  the  serum 
and  the  urine  and  comparing  them.  The  method  is  known 
as  cryoscopy,  and  depends  upon  the  fact  that  the  lowering 
of  the  freezing  point  of  a  liquid  varies  directly  with  the 
amount  of  salts  in  solution.  The  method  is  a  long  and 
tiresome  one  to  perform.  In  the  other  method  the  electrical 
conductivity  of  a  volume  of  the  serum  is  measured  by  a 
modification  of  the  Wheatstone  bridge.  The  serum  is  replaced 
by  an  equal  volume  of  a  sample  of  urine,  passed  at  a  period 
corresponding  to  the  time  at  which  the  blood  was  drawn, 
and  the  conductivity  of  the  urine  is  measured.  The  ratio 
of  the  conductivity  of  the  serum  to  that  of  the  urine  is  the 
ratio  of  the  inorganic  salts  in  solution  in  these  fluids.  The 
actual  measurement  of  the  conductivity  is  simple  and  takes 
a  few7  minutes  only.  A  special  and  somewhat  expensive 
apparatus  is  required,  and  a  fairly  extensive  trial  has  not 
convinced  me  that  the  test  provides  any  real  clinical 
information  such  as  cannot  be  obtained  by  a  careful 
examination  of  the  patient  and  his  urine  by  ordinary 
methods. 

Peoteids  in  the  Urine. 

The  routine  tests  for  the  presence  of  albumin  in  the  urine 
have  already  been  given.  When  albumin  is  present  the 
amount  should  always  be  stated,  and  for  all  practical 
purposes  it  is  sufficient  in  nearly  all  cases  to  measure  the 
amount  by  the  following  rough  scheme.  If  a  cloud  only 
appears,  such  as  would  cause  no  appreciable  bulk  of  precipitate 
at  the  bottom  of  the  test  tube,  note  that  a  faint  trace,  a 
trace,  or  a  heavy  cloud  of  albumin  is  present  according  to 
the  opacity  which  is  produced  after  boiling  and  acidifying. 


238  CLINICAL   PATHOLOGY. 

If  a  precipitate  is  formed  in  bulk,  boil  the  whole  contents  of 
the  test-tube  and  allow  it  to  stand.  When  the  precipitate 
has  all  collected  at  the  bottom,  hold  an  ordinary  tape 
measure  against  the  test  tube,  and  read  the  level  of  the 
precipitate  and  of  the  urine.  Express  the  depth  of  the  precipi- 
tate as  a  fraction  of  the  depth  of  the  column  of  urine  as  one- 
sixth,  one-fourth,  one-third,  etc.  Should  a  slightly  more 
accurate  method  be  required,  Esbach's  albuminometer 
can  be  used.  This  instrument  does  not  measure  traces  of 
albumin,  and  if  very  much  albumin  is  present  it  is  necessary 
to  dilute  the  urine  and  to  allow  for  the  dilution  after  taking 
the  reading.     To  use  the  apparatus  : — 

Fill  the  tube  with  the  urine  (which  should  be  acidified  if 
necessary)  up  to  the  mark  U. 

Pour  in  the  reagent  up  to  the  mark  E. 

Cork  the  tube  and  mix  by  inverting  several  times. 

Allow  to  stand  for  24  hours. 

Bead  the  level  of  the  precipitate. 

The  scale  gives  the  grammes  of  proteid  present  in  a  litre  of 
urine. 

The  percentage  is  consequently  obtained  by  dividing  by  10. 

The  reagent  has  the  following  composition : — 

Picric  acid        ....         10  grammes. 
Citric  acid         .         .         .         .         20         ,, 
Water 1  litre. 

The  method  is  not  remarkably  accurate,  since  the  space 
occupied  by  the  precipitate  continues  to  vary  with  the  time 
the  tube  is  allowed  to  stand,  and  all  albuminous  precipitates 
do  not  appear  to  take  the  same  time  to  settle.  The  percentage 
of  proteids,  however,  is  given  with  sufficient  accuracy  for 
clinical  purposes,  and  the  exact  estimation  of  proteids  in  the 
urine  is  a  tedious  process  and  conveys  no  further  information 
of  practical  value. 

The  nature  of  coagulable  proteids  in  the  urine  is  of  no 
particular  clinical  significance,  and  they  consist  as  a  rule  mainly 
of  serum  albumin  with  a  lesser  amount  of  serum  globulin. 

The  amounts  of  serum  albumin  and  serum  globulin  can  be 
estimated  as  follows  : — 

Bender  about  200  c.c.  of  urine  slightly  alkaline  with 
ammonia. 

Filter. 


VARIATIONS   IN   ACIDITY   AND   ACIDOSIS,   ETC.     239 

To  100  c.c.  of  the  filtrate  add  solid  ammonium  sulphate  to 
saturation. 

Allow  to  stand  overnight. 

The  total  proteids  are  precipitated. 

Filter  off  the  precipitate  through  a  weighed  ash-free 
paper. 

Dry  and  weigh. 

Incinerate  and  weigh  the  ash. 

Deduct  the  weight  of  the  ash ;  the  difference  gives  the  total 
proteids. 

To  another  100  c.c.  of  the  filtered  urine  add  100  c.c.  of  a 
saturated  solution  of  ammonium  sulphate. 

Proceed  as  before. 

The  precipitate  contains  the  globulins,  and  the  difference 
between  the  two  precipitates  may  be  reckoned  as  albumin. 

The  following  are  among  the  more  important  conditions  in 
which  albumin  occurs  in  the  urine  : — 

Albumin  may  occur  after  violent  exercise,  severe  exposure, 
and  after  excessive  eating  or  drinking.  In  the  previously 
normal  individual  the  albuminuria  which  may  be  induced  by 
such  means  rapidly  disappears.  Fever,  particularly  if 
considerable  or  prolonged,  is  commonly  associated  with  a 
trace  of  albumin.  In  chronic  interstitial  nephritis  albumin 
is  present  only  as  a  trace  so  long  as  cardiac  compensa- 
tion lasts.  In  diabetes  there  is  commonly  a  trace  of 
albumin. 

Albumin  in  large  amount  is  present  in  acute  nephritis 
of  all  varieties,  including  the  nephritis  of  eclampsia  and 
chronic  parenchymatous  nephritis,  also  in  amyloid  disease  of 
the  kidney  and  in  cardiac  failure  with  partial  suppression 
of  urine. 

In  cases  of  albuminuria  due  to  any  form  of  renal  disease 
casts  are  nearly  always  present  in  addition. 

Albuminuria  may  also  occur  and  in  considerable  degree 
without  any  known  structural  alteration  in  the  kidneys  or 
heart.  Such  cases  are  known  as  functional  or  postural  albu- 
minuria, and  are  not  uncommon  in  young  people.  Such 
albuminuria  tends  to  disappear  when  the  patient  is  at  rest 
and  to  reappear  on  taking  exercise  or  even  on  assuming  the 
erect  posture.  Casts  are  absent  and  the  arteries  are  not 
affected,  nor  do  these  cases  commonly  appear  to  progress  to 


240  CLINICAL   PATHOLOGY. 

any  of  the  recognised  forms  of  nephritis.  Albuminuria  with- 
out renal  or  vascular  degeneration  may  likewise  follow  one  of 
the  infectious  fevers. 

The  occurrence  of  blood  or  pus  in  the  urine  is  always 
associated  with  the  presence  of  albumin,  and  it  is  frequently 
of  importance  to  determine  whether  the  amount  of  albumin  is 
such  as  could  be  accounted  for  by  the  quantity  of  pus  or  blood 
or  is  in  excess  of  it.  Albumin  present  in  greater  amount  than 
can  be  accounted  for  by  the  amount  of  pus  coming  from  a 
probable  focus  in  the  bladder,  for  example,  would  point  to 
involvement  of  the  kidneys  as  well.  It  may  be  taken  as  a 
rough  guide  that  a  very  considerable  amount  of  pus  in  the 
urine  produces  little  more  than  a  trace  of  albumin.  The 
presence  of  blood  leads  to  a  higher  degree  of  albuminuria  than 
the  same  amount  of  pus,  and  hematuria  occurring  with  a 
contracted  granular  kidney  obscures  the  albumin  due  to  the 
nephritis.  In  such  cases  the  presence  of  granular  casts  would 
indicate  renal  involvement.  The  hEematuria  which  accom- 
panies acute  nephritis  is  usually  associated  with  a  degree  of 
albuminuria  obviously  much  in  excess  of  the  amount  of  blood 
present. 

The  exact  significance  to  be  attached  to  the  presence  of 
albumin  in  the  urine  is  of  great  importance  in  life  insurance, 
and  every  case  in  which  even  a  trace  is  discovered  calls  for  a 
very  complete  examination,  not  only  of  the  urine  but  of  the 
patient  also.  There  is  no  doubt  that  transient  albuminuria 
may  occur  without  any  serious  involvement  of  the  kidney,  and 
in  exceptional  cases  a  considerable  percentage  of  albumin  may 
be  passed  over  long  periods  without  any  apparent  detriment 
to  the  individual. 

Proteoses  may  be  present  in  fevers,  in  long-standing  cases 
of  suppuration,  and  rarely,  if  ever,  in  nephritis ;  they  may  be 
tested  for  as  follows : — 

Saturate  the  urine  with  ammonium  sulphate. 

Heat  to  coagulate  the  proteins  which  may  be  present. 

Filter  and  extract  the  precipitate  with  alcohol  to  remove  the 
urobilin  which  gives  the  biuret  reaction. 

Extract  precipitate  with  boiling  water,  which  dissolves  the 
proteoses. 

Test  the  solution  by  the  biuret  reaction  and  with  Millon's 
reagent. 


VARIATIONS    IN   ACIDITY  AND   ACIDOSIS,   ETC.     241 

The  biuret  reaction  is  performed  as  follows  : — 

To  the  aqueous  solution  add  caustic  soda  and  2  drops  of  a 
1  per  cent,  solution  of  copper  sulphate. 

A  pink  colour  is  produced. 

Millon's  reagent  consists  of  mercury  dissolved  in  concen- 
trated nitric  acid. 

Add  the  reagent  to  the  solution  and  heat. 

A  deep  red  coloration  is  produced. 

The  examination  of  the  urine  for  proteoses  is  only  occasion- 
ally called  for.  There  is,  however,  an  extremely  rare  disease 
of  the  bone  marrow  in  which  almost  the  entire  marrow  is 
transformed  into  a  sarcoma-like  substance,  and  in  these  cases 
an  abundant  proteosuria  is  present  and  is  pathognomonic  of 
the  disease.  The  exact  nature  of  the  protein,  which  is  called 
the  Bence-Jones  protein,  is  not  known,  but  it  is  readily 
recognised  in  the  urine  by  the  following  simple  test : — 

Filter  the  urine  into  a  test  tube. 

Place  the  test  tube  in  a  water  bath  at  about  45°  C. 

Slowly  raise  the  temperature  of  the  water. 

When  the  temperature  rises  to  between  50°  and  60°  C.  the 
urine  becomes  turbid  from  the  coagulated  proteid. 

On  raising  the  temperature  still  further  the  turbidity  lessens 
and  finally  disappears  entirely. 

On  cooling  the  urine  again  the  precipitate  returns. 

Mucin  is  not  infrequently  present  in  the  urine  in  associa- 
tion with  pus,  and  in  connection  with  numerous  crystalline 
deposits.  The  presence  of  mucin  out  of  solution  is  of  no 
particular  significance  except  that  it  may  be  mistaken  on 
naked-eye  examination  for  pus. 

When  the  mucin  is  in  solution  it  may  be  detected  by  dilut- 
ing the  urine  with  an  equal  volume  of  water  and  adding  a  few 
drops  of  acetic  acid,  when  the  mucin  is  precipitated  as  a  white 
cloud  insoluble  in  excess  of  acid. 

Carbohydrates  in  the  Urine. 

Glucose  is  the  most  important  carbohydrate  which  may  be 
present  in  the  urine,  and  tests  for  its  detection  form  part  of 
the  routine  examination  of  all  specimens.  The  qualitative 
tests  for  glucose  have  already  been  given.  Should  glucose  be 
present  its  amount  must   always   be   estimated.     There  are 

p.  16 


242  CLINICAL   PATHOLOGY. 

numerous  quantitative  methods  in  common  use,  and  the 
following  can  be  recommended  as  simple  and  accurate.  It  is 
known  as  Gerrard's  method. 

Gerrard's  method  depends  upon  the  fact  that  the  colourless 
(or  almost  colourless)  double  cyanide  of  potash  and  copper 
dissolves  the  coloured  cuprous  oxide  precipitate  as  it  is  formed 
in  Fehling's  solution.  The  estimation  is  performed  as 
follows  : — 

Dilute  the  urine  according  to  the  intensity  of  the  Fehling's 
reaction  obtained.  Thus,  if  an  abundant  precipitate  resulted 
on  the  addition  of  a  few  drops  of  boiling  urine,  dilute  the 
urine  10  times ;  in  cases  of  moderate  glycosuria  a  dilution  of 
5  times  is  sufficient. 

The  dilution  to  be  aimed  at  is  one  in  which  from  10  to  15  c.c. 
of  the  diluted  urine  reduce  10  c.c.  of  Fehling's  solution.  If 
the  first  dilution  is  unsuitable  another  can  be  made.  All 
dilutions  must  be  measured  accurately  and  can  be  made  with 
tap  water. 

Einse  out  a  burette  with  a  few  cubic  centimetres  of  the 
diluted  urine  before  filling. 

Measure  out  carefully  with  a  delivery  pipette  10  c.c.  of 
Fehling's  solution  and  expel  into  a  porcelain  evaporating  dish. 

Add  about  BO  c.c.  of  Gerrard's  solution. 

Bring  to  the  boil  over  the  flame. 

Eun  in  the  diluted  urine  from  the  burette. 

See  that  the  mixture  is  boiling  all  the  time  and  keep  it 
stirred  with  a  glass  rod. 

The  reaction  is  complete  when  the  last  trace  of  blue  colour 
has  gone  from  the  mixture. 

The  colour  partially  returns  on  cooling. 

The  titration  should  be  performed  as  rapidly  as  possible. 

The  calculation  is  made  as  follows  : — 

10  c.c.  of  Fehling's  solution  =  '05  gramme  glucose. 

If  20  c.c.  of  urine  diluted  1  in  10  have  been  used,  then  the 

actual  amount  of  urine  needed  was  2  c.c,  and  2  c.c.  of  urine 

contain  "05  gramme  glucose,  therefore  100  c.c.  of  urine  contain 

•05    X    100       0  K 
or  2*5  grammes. 

Knowing  the  total  measurement  of  the  urine,  the  output 
of  glucose  for  the  24  hours  can  be  calculated  from  the 
percentage. 


VARIATIONS    IN    ACIDITY   AND   ACIDOSIS,   ETC.     243 

The  Gerrard's  solution  used  is  not  concerned  in  the  calcula- 
tion, since  the  cyanide  undergoes  no  reduction. 

Gerrard's  solution  is  prepared  as  follows : — 

Dilute  100  c.c.  of  Fehling's  solution  with  300  c.c.  of  water. 

Boil. 

Run  in  a  5  per  cent,  solution  of  potassium  cyanide  until 
the  blue  colour  has  gone. 

Make  up  the  mixture  to  500  c.c.  with  water. 

The  solution  may  remain  a  very  faint  blue,  but  it  should  be 
as  free  from  colour  as  possible.     It  will  keep  for  several  weeks. 

Glucose  is  present  in  the  urine  under  the  following  con- 
ditions : — 

The  urine  of  untreated  diabetics  usually  contains  glucose 
in  considerable  amount,  and  is  of  a  high  specific  gravity  and 
large  quantity.  The  "alimentary"  glycosuria  of  middle- 
aged  or  older  persons  is  largely  dependent  upon  the  intake 
of  carbohydrates.  On  a  strict  carbohydrate-free  diet  the 
glucose  commonly  disappears  from  the  urine,  and  such 
persons  on  a  comparatively  mixed  diet  may  continue  to 
excrete  a  moderate  amount  of  glucose  for  years  and  live  to 
an  old  age.  The  diabetes  of  younger  people,  that  is  of 
an  age  less  than  25,  almost  invariably  runs  a  rapidly 
fatal  course  ending  in  coma.  The  glucose  in  the  urine  of 
such  cases  is  much  less  affected  by  diet  than  is  the  glycosuria 
of  old  persons.  The  rapidly  fatal  form  of  diabetes  may, 
however,  occur  at  any  age. 

In  addition  to  diabetes  proper,  glucose  may  occur  in  the 
urine  in  a  considerable  variety  of  affections,  but  in  almost 
every  instance  the  glucose  is  in  small  or  comparatively  small 
amount,  is  transitory,  and  is  not  associated  w7ith  the  symptoms 
of  diabetes.  In  diabetes  the  glycosuria  is  the  most  striking- 
sign  of  the  disease,  wThile  in  other  affections  the  presence  of 
glucose  in  the  urine  is  an  interesting  but  casual  phenomenon 
only. 

Glycosuria  of  this  nature  may  arise  under  the  following 
conditions  : — 

Alimentary  disturbances,  such  as  starvation  or  the  inges- 
tion of  excessive  carbohydrate  meals. 

Toxic  causes,  in  particular  after  the  taking  of  morphine, 
strychnine,  phloridzin,  or  phosphorus,  and  during  acute  fevers. 

Nervous  diseases    such  as  haemorrhage  into  the  pons. 

16—2 


244  CLINICAL   PATHOLOGY. 

cerebral    tumour   or   cyst,    meningitis,   syphilitic    and   para- 
syphilitic  diseases  of  the  central  nervous  system. 

Pregnancy  may  be  associated  with  a  glycosuria  as  well  as  a 
lactosuria. 

Diseases  of  the  secreting  glands,  as  of  the  thyroid  in 
exophthalmic  goitre  and  myxoedema,  of  the  pituitary  gland 
in  acromegaly,  of  the  pancreas  in  acute  or  chronic  pancreatitis 
or  in  tumours  of  the  pancreas. 

Renal  affections,  of  which  phloridzin  diabetes  is  the  best 
known  type. 

Hepatic  diseases,  as  in  cirrhosis  of  the  liver  and  in  gall 
stones. 

In  all  cases  in  which  a  reducing  substance  is  present  in  the 
urine  its  nature  should  be  determined,  and  if  the  substance 
proves  to  be  glucose  its  amount  should  be  estimated.  The 
investigation  does  not,  however  stop  at  this  point.  It  is 
necessary  to  discover  if  the  patient  has  the  symptoms  and 
signs  of  diabetes  or  if  he  is  suffering  from  some  other  obvious 
affection  known  to  be  occasionally  associated  with  glycosuria. 
In  all  diabetic  cases  the  effect  of  diet  on  the  output  of  glucose 
has  to  be  d^  termined  over  a  series  of  observations.  A  further 
and  important  examination  of  the  urine  has  also  to  be  made 
in  addition  to  the  ordinary  routine  processes.  The  urine  has 
to  be  examined  for  evidence  of  acidosis.  The  abnormal 
substances  which  may  be  present  in  the  urine  are 
B-hydroxybutyric  acid,  aceto-acetic  acid  and  acetone. 
The  B-hydroxybutyric  acid  is  the  mother  substance,  which 
on  oxidation  yields  aceto-acetic  acid  and  water. . 
CU3  •  CH  (OH)  CH2 .  COOH  +  0  = 
CH3 .  CO.  CH2 .  COOH  +  H20. 

The  aceto-acetic  acid  is  readily  decomposed  into  acetone 
and  C02 . 

CH3.  CO.  CH2 .  COOH  =  CH3 .  COCH3.  +  C02. 

The  substances  to  be  tested  for  in  the  urine  are  acetone  and 
aceto-acetic  acid.  If  these  are  present  B-hydroxybutyric  acid 
may  be  presumed. 

The  following  test  for  aceto-acetic  acid  should  be 
employed : — 

Gerhardt's  reaction. — Filter  fresh  unboiled  urine. 

Add  dilute  ferric  chloride  drop  by  drop  until  no  more 
precipitate  forms. 


VARIATIONS   IN   ACIDITY   AND   ACIDOSIS,  ETC.     245 

Filter  and  add  a  few  more  drops  of  the  ferric  chloride. 

A  claret-red  colour  is  produced,  which  disappears  on 
prolonged  heating. 

Certain  drugs,  such  as  antipyrin  or  salicylates,  give  a  similar 
colour,  which,  however,  persists  on  heating. 

If  the  acid  is  present  in  considerable  amount  it  is  sufficient 
to  add  4  or  5  drops  of  the  ferric  chloride  to  the  urine,  an 
obvious  deep  red  colour  resulting.  The  colour  does  not 
always  disappear  completely  on  boiling. 

Acetone  in  the  urine  and  in  the  patient's  breath  may  be 
recognised  by  its  characteristic  "  fruity  "  odour.  The  odour 
of  acetone  is  more  readily  recognised  by  some  individuals 
than  by  others,  and  many  persons  are  quite  unable  to  detect  it 
by  the  smell  unless  it  be  present  in  exceptional  amount.  The 
peculiar  odour  of  typhoid  patients  is  in  a  similar  way  very 
characteristic  to  some  observers. 

The  test  for  acetone  which  depends  upon  its  conversion 
into  iodoform  is  not  very  satisfactory,  and  the  following  test 
is  preferable: — 

Rothera's  test.— To  5  c.c.  of  urine  add  3  grammes  of 
ammonium  sulphate. 

Add  3  drops  of  a  freshly-prepared  solution  of  sodium 
nitro-prusside. 

Add  2  c.c.  of  ammonia. 

Acetone  gives  a  slowly  developing  permanganate  colour. 

The  presence  of  these  bodies  in  the  urine  of  diabetic 
patients  is  of  considerable  importance,  since  the  onset  of 
coma  is  associated  with  their  occurrence.  In  cases  of 
glycosuria,  which  can  be  cured  by  appropriate  diet,  acetone 
and  diacetic  acid  are  almost  invariably  absent :  their  presence 
is  therefore  indicative  of  the  graver  form  of  diabetes  and  of 
the  onset  of  coma.  Exceptional  cases  are,  however,  met  with 
in  which  these  bodies  may  be  present  at  intervals  for  many 
years  without  grave  symptoms. 

Diacetic  acid  is  also  found  in  the  other  conditions  which 
have  previously  been  mentioned  as  being  associated  with 
acidosis.  The  "  acidity  "  of  the  urine  also,  as  previously  de- 
scribed, should  be  periodically  investigated  in  cases  of  true 
diabetes.  An  abnormal  acidity  is  evidence  of  an  abnormal 
metabolism  of  the  body  tissues,  whether  of  proteids  or  fats,  and 
the  ratio  of  the  ammonia  nitrogen  is  in  reality  of  considerably 


246  CLINICAL   PATHOLOGY. 

more  clinical  significance  than  the  amount  of  glucose  in  the 
urine. 

Lactose  is  not  infrequently  found  in  the  urine  of  pregnant 
or  nursing  women.  Its  occurrence  is  of  no  particular  import- 
ance except  that  it  is  apt  to  be  mistaken  for  glucose.  Lactose 
has  a  similar  reducing  action  upon  Fehling's  solution  to 
glucose. 

Both  lactose  and  glucose  yield  osazone  crystals.  The  form 
of  the  crystals  in  the  two  cases  is,  however,  different. 
Glucosazone  crystals  are  long  feathery  sheaves ;  the 
lactosazone  form  more  circular  bunches  of  spidery  crystals 
(see  plate). 

Lactose  is  not  fermented  by  yeast,  and  by  this  test  is  readily 
differentiated  from  glucose. 

The  osazone  reaction  in  the  urine  is  obtained  as 
follows  : — 

Place  about  50  c.c.  of  urine  in  a  beaker. 

Add  1  gramme  of  sodium  acetate. 

Add  *5  gramme  of  phenyl  hydrazin  hydrochlorate. 

Stir  well  and  place  on  a  water  bath. 

Leave  for  one  hour. 

Allow  to  cool  slowly  at  room  temperature. 

Examine  a  drop  of  the  deposit  (after  centrifuging  at  a  low 
speed  if  necessary)  on  a  slide  beneath  a  cover-slip  and  with 
the  low  power  of  the  microscope. 

The  test  is  rarely  successful  when  only  very  small  quan- 
tities of  carbohydrate  are  present. 

Pentose  may  occasionally  be  met  with  in  the  urine. 

Urines  which  contain  pentose  reduce  Fehling's  solution  and 
yield  an  osazone  crystal.    Pentose  does  not  ferment  with  yeast. 

Pentoses  give  the  following  tests  with  phloroglucinol  and 
orcinol : — 

Phloroglucinol  reaction.  —  Mix  equal  parts  of  con- 
centrated hydrochloric  acid  and  water  in  a  test  tube. 

Add  a  little  phloroglucinol. 

Add  5  to  10  drops  of  urine. 

Warm  in  the  water  bath. 

The  solution  gradually  becomes  cherry  red  and  a  precipitate 
forms. 

Allow  to  cool. 

Shake  with  amyl  alcohol. 


PLATE   XL 


Glucosazone  Crystals.  Lactosazone  Crystals. 


Pancreatic  Test  C  "  Crystals.  Uric  Acid  Crystals. 


PLATE    XI. 


O  0 


o 


VARIATIONS   IN   ACIDITY   AND   ACIDOSIS,   ETC.     247 

Examine  the  red  amyl  alcohol  solution  with  the  spectroscope. 

The  spectrum  shows  an  absorption  band  between  D  and  E. 

Orcinol  reaction. — This  reaction  is  performed  in  exactly 
the  same  manner.  The  solution  in  the  water  bath  becomes 
first  red,  then  violet,  and  finally  blue.  The  amyl  alcohol 
extract  is  bluish  green  and  shows  an  absorption  band  between 
C  and  D. 

The  same  reactions  are  given  with  glycuronic  acid,  which 
may  appear  in  the  urine  in  considerable  amount  after  the 
administration  of  certain  drugs,  such  as  chloral,  chloroform 
and  morphia.  Occasionally  it  occurs  spontaneously  in  the 
urine.  Urines  containing  glycuronic  acid  give  much  the  same 
reactions  as  those  containing  pentose.  Glycuronic  acid  is 
optically  active ;  the  free  acid  is  dextro-rotatory,  and  the 
combined  acid,  which  alone  occurs  in  the  urine,  is  Iebvo- 
rotatory. 

The  presence  of  these  substances  in  the  urine  is  of 
importance,  since  they  may  be  mistaken  for  glucose.  The 
occurrence  of  pentose  in  the  urine  is  of  considerable  scientific 
interest  in  that  it  may  occur  in  apparently  healthy  persons 
as  a  notable  example  of  one  of  the  rare  inborn  errors  of 
metabolism. 


CHAPTER  XVIII. 

urinary  deposits — urinary  calculi. 

Organised  Urinary  Deposits. 

The  examination  of  urinary  deposits  with  the  microscope  is 
more  important  and  more  neglected  than  almost  any  other 
laboratory  investigation.  The  student  working  in  the  wards 
is  more  concerned,  and  rightly,  with  the  physical  signs  and 
symptoms  of  the  patient ;  consequently  he  prefers  to  content 
himself  with  a  note  of  the  naked-eye  appearance  of  a  deposit, 
or  at  the  most  with  a  rough  chemical  test.  The  recognition 
of  the  various  formed  elements  which  may  be  present  in  the 
urine  requires  some  practice,  and  this  is  more  conveniently 
acquired  in  a  laboratory  than  in  the  wards.  The  time  available 
in  the  wards  is  limited,  and  the  ward  microscope,  however 
excellent  an  instrument  it  may  have  been  in  its  youth,  is  apt 
to  reflect  more  light  from  its  brass  work  than  it  admits  through 
its  lenses.  Fortunately  for  those  students  and  practitioners 
who  have  been  unable  to  avail  themselves  of  a  laboratory 
course,  an  acquaintance  with  urinary  deposits  is  fairly  readily 
acquired  without  personal  instruction.  The  deposits  particu- 
larly lend  themselves  to  accurate  reproduction  by  diagram, 
and  are  among  the  minority  of  pathological  objects  which  can 
be  recognised  by  reference  to  a  plate.  An  attempt  is  made 
here  to  describe,  and  so  far  as  possible  illustrate,  such  deposits 
as  are  likely  to  be  met  with  in  the  urine.  Those  who  require 
a  wider  knowledge  of  the  subject  are  referred  to  the  excellent 
text-book  of  Rieder  and  Delepine. 

The  methods  of  examining  a  urinary  deposit  may  be  again 
recapitulated.  If  a  considerable  deposit  is  present  in  the 
specimen  glass  do  not  centrifuge.  Draw  up  a  portion  of  the 
deposit  in  a  pipette.  If  the  deposit  is  scanty  or  absent,  wash 
out  thoroughly  two  centrifuge  tubes.  Shake  up  the  bottom 
portion  of  the  specimen  after  carefully  pouring  off  the  super- 
natant fluid.     Fill  the  centrifuge  tubes  with  urine  and  centri- 


UKINARY  DEPOSITS— URINARY   CALCULI.      249 

fuge  for  a  few  minutes  at  a  moderate  speed.  Do  not  stop  the 
centrifuge  after  turning  off  the  power,  but  allow  it  to  run  down. 
Remove  the  tubes  and  make  use  of  the  one  that  contains  the 
more  deposit.  Turn  the  tube  upside  down  :  the  bulk  of  the 
deposit  will  remain  in  the  last  drop,  which  will  not  fall  out, 
Prepare  a  clean  slide  and  cover-glass.  Shake  out  a  drop  of  the 
urine  containing  the  deposit  on  the  centre  of  the  slide.  Let 
down  a  cover -glass  on  the  slide.  If  the  right  amount  of 
deposit  is  taken  there  will  be  no  air  bubbles  and  the  urine  will 
not  spread  over  the  slide  beyond  the  cover-glass.  Examine 
with  the  microscope  vertical  and  the  diaphragm  partly  closed. 
Use  both  a  f-inch  and  a  ^-inch  objective. 

Blood. — Red  blood  corpuscles  are  recognised  under  the 
higher  objective  by  their  shape,  size  and  colour.  The  size  and 
shape  are  not  infrequently  altered  by  crenation ';  the  colour  is 
very  distinctive.  With  a  little  practice  a  single  red  cell  can  be 
recognised  with  certainty.  The  objects  most  frequently  con- 
founded with  red  blood  corpuscles  are  oil  globules  and  uric 
acid  crystals.  Oil  globules  are  often  seen  in  the  deposits  of 
catheter  specimens,  and  are  derived  from  the  lubricant  used 
for  the  catheter.  They  are  more  circular  than  red  cells  and 
of  very  varying  sizes  from  circles  smaller  than  red  cells  to 
obvious  large  globules.  They  are  yellowish  in  colour,  but  of  a 
different  tint  from  the  corpuscles.  Uric  acid  crystals  mislead 
only  by  their  colour ;  they  can  be  recognised  by  their  regular 
outlines  and  crystalline  shape. 

Should  there  be  any  doubt  as  to  the  nature  of  the  corpuscles, 
a  film  preparation  should  be  made  and  stained  with  Leishman's 
stain. 

Blood  may  also  appear  in  the  urine  as  a  contamination 
from  some  other  part  of  the  body,  and  in  cases  of  doubt, 
particularly  in  specimens  obtained  from  women,  a  catheter 
specimen  should  be  examined. 

The  presence  of  blood  in  the  urine  will  probably  have  been 
confirmed  by  the  guiac  test  or  by  the  spectroscope.  The 
amount  of  blood  which  can  be  detected  by  the  microscope 
may  be  so  small  as  to  escape  observation  by  the  other  tests. 

In  all  cases  of  hematuria  an  estimate  should  be  made  of 
the  relative  proportions  of  red  cells  and  haemoglobin  present. 
There  is  no  difficulty  in  this.  If  sufficient  blood  is  present 
to  obviously  colour  the  urine  a  very  large  number   of   red 


250  CLINICAL   PATHOLOGY. 

cells  will  be  present  in  the  deposit.  In  cases  of  marked 
hemoglobinuria  red  cells  will  be  almost  entirely  absent  or 
very  scanty  and  very  distorted. 

Pus  (Plate  XII.). — A  curious  convention  has  grown  up  about 
the  meaning  of  the  phrase  "  pus  corpuscles."  The  student  has 
been  led  to  look  for  some  new  and  strange  pathological  entity. 
Pus  corpuscles  are  phagocytic  cells,  and  as  such  consist  of 
polynuclear  neutrophils  with  occasional  large  hyaline  cells  and 
epithelial  cells.  It  is  true  that  the  corpuscles  may  be  more  or 
less  degenerated,  but  the  majority  of  cells  in  most  samples  of 
pus  are  perfectly  well  formed  and  normal  in  appearance. 
There  is  no  actual  dividing  line  between  a  polynuclear  cell 
and  a  pus  corpuscle.  If  polynuclear  cells  are  sufficiently 
numerous  to  produce  a  naked-eye  deposit,  pus  is  present.  If 
few  cells  are  found  the  process  is  the  same,  and  the  difference 
is  one  of  degree  only. 

Urines  containing  pus  cells  in  large  numbers  give  a  greenish 
colour  with  the  guiac  test,  but  the  microscope  is  the  only 
reasonably  accurate  method  of  detecting  pus.  The  perform- 
ance of  the  potash  test  for  pus  is  a  mere  waste  of  time  and  is 
not  described  here. 

Pus  corpuscles  are  readily  recognised  in  unstained  specimens 
by  their  size,  their  round  shape,  the  commonly  bilobed  or  poly- 
nuclear form  of  their  nuclei,  and  by  their  refractile  granular 
appearance. 

"When  a  considerable  degree  of  pyuria  is  present  the  pus  may 
be  partially  obscured  by  the  presence  of  "  mucus  "  in  addition. 
The  pus  in  such  cases  is  very  viscid  and  difficult  to  control 
when  preparing  a  microscopic  specimen.  When  viewed  under 
the  microscope  the  cells  are  partially  hidden  under  the 
"  mucus"  and  can  only  be  seen  by  careful  focussing.  Such 
specimens  floated  in  water  may  resemble  slimy  membranes  in 
appearance  and  may  have  the  shape  of  the  bladder  or  urethra. 
Prostatic  casts  are  of  similar  composition. 

In  cases  of  doubt  a  thin  film  should  be  spread  on  a  slide 
with  a  platinum  wire,  dried,  stained  for  3  minutes  with  carbol- 
thionin,  washed  in  water,  blotted  dry,  and  examined  with  an 
immersion  lens  or  even  with  a  ^t-inch  objective  after  mounting 
in  Canada  balsam.  The  polynuclear  cells  are  often  somewhat 
shrunken  in  such  specimens,  but  are  perfectly  recognisable,  and 
in  addition  any  bacteria  which  may  be  present  can  be  noted. 


PLATE    XII. 


®m@>^^ 


© 


® 


® 


# 
® 


© 


©      ® 


M &_ 


0 


Pus  Corpuscles. 
(Urinary  Deposit.) 


Epithelial  Cells. 
(Urinary  Deposit.) 


-".   .  ®         (A    o 


O  ® 

.      O 


Sfe       ©      ^^^ 


'  *i°».  »*•'■ 


0 


® 
o 


§J«fe.  O 


Casts,  etc. 
(From  the  Deposit  of  a  Case  of  Acute  Nephritis.) 


f 

\ 

^■f 

* 

•*^ 

Tyrosine  Crystals. 
(From  Urine,  after  Separation.) 


Spermatozoa. 

(From  a  Spermatocele.) 

(Stained  Specimen.) 


URINARY  DEPOSITS— URINARY   CALCULI.      251 

It  is  occasionally  necessary  to  determine  if  a  urine  which 
contains  blood  has  pus  present  in  addition,  since  polynuclear 
cells  are  necessarily  present  when  the  bleeding  has  been 
free.  The  question  is  fairly  easily  answered  by  the  micro- 
scopic examination  of  the  deposit.  In  the  case  of  blood  the 
average  field  of  the  ^-inch  objective  shows  large  numbers  of 
red  cells  and  1  or  at  most  2  or  3  leucocytes.  If  pus  is  present  in 
addition  the  field  will  show  a  dozen  or  more  leucocytes  among 
the  red  cells,  and  fields  will  be  found  in  which  the  leucocytes 
are  gathered  in  small  clusters.  Conversely  in  cases  of  con- 
siderable pyuria  occasional  red  cells  will  usually  be  detected 
among  the  leucocytes. 

The  presence  of  polynuclear  leucocytes  in  a  urinary  deposit 
is  pathological,  provided  that  they  came  from  some  part  of  the 
urinary  tract.  It  is  essential  for  the  detection  of  small  traces 
of  pus  in  the  urine  of  women  that  a  catheter  specimen  should 
be  examined.  In  men  the  pus  may  come  from  the  urethra 
in  cases  of  gonorrhoea,  and  it  is  not  infrequent  to  find  a  few 
leucocytes,  probably  derived  from  the  prostatic  urethra,  in  the 
urine  of  patients  who  have  had  gonorrhoea  many  years 
previously.  The  pus  in  such  cases  may  or  may  not  be  due  to 
the  actual  presence  of  gonococci.  The  pus  cells  are  frequently 
united  by  "mucus"  into  long  thread-like  processes  visible  to 
the  naked  eye  and  known  as  prostatic  threads.  These  threads 
are  most  often  present  in  the  first  specimen  of  urine  passed 
in  the  morning,  and  may  be  seen  best  after  massage  of  the 
prostate. 

Epithelium  (Plate  XII.).  —  Epithelial  cells  are  com- 
monly present  in  the  urine,  and  in  deposits  taken  from  the 
urine  of  female  patients  are  as  a  rule  very  abundant. 
Epithelial  cells  in  the  urine  have  no  pathological  significance, 
but  must  be  recognised,  since  they  are  apt  to  be  mistaken  for 
pus  corpuscles,  or  even  portions  of  new  growth. 

Epithelial  cells  may  be  recognised  by  their  size,  being  com- 
monly much  larger  than  the  polynuclear  cells,  by  their  shape, 
which  is  rarely  round,  by  the  absence  of  granularity,  and  by 
their  single  central  nucleus.  They  cannot  be  distinguished 
from  the  single  cells  of  a  neoplasm. 

Epithelial  cells  differ  considerably  in  shape  and  size,  and 
the  student  is  commonly  asked  to  recognise  from  the  nature 
of  the  cell  that  portion  of  the  urinary  tract  from  which  it  is 


252  CLINICAL   PATHOLOGY. 

derived.  I  am  by  no  means  convinced  that  this  is  possible. 
The  commonest  form  of  epithelial  cell  is  a  very  large  angular 
cell  of  the  squamous  type.  It  is  the  predominant  cell  in  the 
urine  of  women  and  may  be  entirely  derived  from  the  vagina. 
Smaller  pear-shaped  and  tailed  cells  are  often  predominant  in 
ureteric  specimens,  and  less  commonly  small  round  cells  which 
closely  resemble  polynuclears,  but  differ  from  them  in  having 
a  round  nucleus.  Such  cells  are  presumably  derived  from  the 
ureter  or  pelvis  of  the  kidney.  The  ureteric  lining  is  very 
friable  and  readily  damaged,  and  specimens  obtained  by 
ureteric  catheterisation  are  frequently  grossly  contaminated  by 
blood.  It  is  only  in  very  skilled  hands  that  epithelial  cells 
only  are  rubbed  off  in  normal,  cases,  and  these  are  commonly 
present  in  abundance  and  have  to  be  carefully  distinguished 
from  pus  corpuscles. 

New  growths. —  In  descriptions  of  the  methods  of 
diagnosing  new  growths  of  the  bladder  or  kidney  the  detec- 
tion of  particles  of  growth  in  the  urine  or  in  the  eye  of 
the  catheter  holds  a  time-honoured  place.  It  is  perfectly 
reasonable  to  look  for  such  particles,  but  it  is  extremely 
rarely  that  one  is  able  to  identify  them.  The  recognition  of  a 
single  cell,  or  even  a  small  cluster  of  cells,  from  a  neoplasm  is 
quite  impossible.  The  more  solid  fragments  in  such  urines 
nearly  always  turn  out  to  be  blood  clots.  Fragments  of 
growth  may,  if  present,  be  recognised  after  teasing  out  if 
necessary,  and  flattening  in  a  drop  of  salt  solution  between 
a  slide  and  cover-slip.  A  drop  of  dilute  methylene  blue  may 
be  allowed  to  run  under  the  cover-slip  if  desired  for  staining 
purposes.  Definite  villus-like  papillary  processes  lined  with 
epithelium  can  only  come  from  a  neoplasm.  Such  processes 
have  to  be  distinguished  from  particles  of  pus  and  epithelial 
cells  bound  together  by  "  mucus."  Pus  of  this  description 
presents  an  undulating  outline,  but  no  regular  processes. 
Actual  naked-eye  fragments  of  a  papilloma  or  carcinoma  are 
in  cases  of  doubt  preferably  fixed  and  sectioned.  The  distinc- 
tion between  a  simple  and  a  malignant  growth  in  such  chance 
particles  may  be  impossible. 

Faecal  elements. — Faecal  elements  may  be  present  in  the 
urine,  particularly  of  female  patients  as  a  chance  contamina- 
tion. The  student  on  examining  the  urinary  deposit  of  a 
catheter  specimen,  and  being  confronted  with  striated  muscle 


URINABY  DEPOSITS -URINARY   CALCULI.      253 

fibres,  may  be  puzzled  to  account  for  them.  Striated  muscle 
fibres  are  yellow  in  colour,  and  show  more  or  less  clearly  the 
transverse  striping.  They  are  quite  unmistakable,  and  in  a 
catheter  specimen  are  definite  evidence  of  abnormal  com- 
munication between  bladder  and  intestine.  The  most 
common  cause  of  such  communication  is  a  recto-vesical 
fistula,  which  may  first  attract  attention  by  the  production  of 
a  cystitis.  If  muscle  fibres  are  absent,  vegetable  fibres  may 
be  recognised  by  their  shape  and  spiral  appearance,  and  in  the 
absence  of  these  elements  faecal  contamination  can  be  inferred 
only  from  the  smell  and  the  general  heterogeneous  appearance 
of  the  deposit. 

Casts  (Plate  XII.).— Casts  are  among  the  most  important 
of  the  formed  elements  in  the  urine.  If  casts  are  present  in 
any  numbers  and  of  certain  varieties^  we  can  be  satisfied  that 
some  form  of  nephritis  is  present,  and  can  sometimes  deter- 
mine which  form.  Tube  casts  are  products  of  the  cells  lining 
the  renal  tubules,  and  their  permanent  form  and  outline  are 
probably  due  to  the  fact  that  they  have  been  formed  in  the 
tubules  and  have  remained  some  time  in  situ.  If  the  tubular 
epithelium  is  normal  and  the  renal  tubules  are  properly 
patent,  no  deposition  of  casts  can  take  place.  The  presence 
of  tubular  casts  in  the  urine  is  consequently  evidence  of  renal 
disease. 

Casts  vary  greatly  in  size,  and  to  a  less  extent  in  shape.  A 
cast  of  average  size  is  just  recognisable  under  a  §-inch  objec- 
tive, and  when  only  a  few  casts  are  present  they  are  best 
searched  for  with  this  power.  A  J-inch  objective  is  necessary 
for  their  verification,  and  should  always  be  used  in  addition. 
Casts  are  recognised  by  the  nature  of  their  contents,  which 
will  be  subsequently  described,  but  particularly  by  their  shape 
and  their  definite  outline.  In  shape  casts  are  long  and  more 
or  less  narrow  cylinders  with  rounded  ends.  Occasionally  one 
end  is  rounded,  the  other  ragged  as  if  fractured.  A  cast 
has  a  clear-cut  and  sharply  defined  outline.  The  limiting 
edge  of  the  cast  distinguishes  it  from  the  only  objects  that 
could  reasonably  be  mistaken  for  it.  Amorphous  urates, 
and  less  commonly  amorphous  phosphates,  may  be  found  in 
little  masses  which  exactly  resemble  casts  in  shape,  and  for 
the  reason  in  some  cases  that  they  are  probably  formed  in  the 
renal  tubules.     Such  urtitic  casts  are  distinguished  in  a  care- 


254  CLINICAL   PATHOLOGY. 

ful  examination  by  the  comparative  irregularity  of  their  edges 
and  the  absence  of  a  definite  outline.  Casts  on  the  whole  are 
remarkably  like  the  representations  of  them  in  many  text- 
books, and  if  the  beginner  sees  a  well-formed  cast  he  almost 
always  recognises  it.  If  he  is  in  doubt  as  to  whether  the 
object  he  sees  is  a  cast  or  not,  it  may  be  anything  from  an 
epithelial  cell  to  a  scratch  on  the  glass,  but  it  is  very  rarely  a 
cast. 

Casts  are  of  several  varieties,  each  of  which  has  a  some- 
what different  significance.  The  casts  are  divided  according 
to  their  structure  into  cellular,  granular,  and  amorphous 
varieties. 

Cellular  casts  are  furthur  subdivided  according  to  the  nature 
of  the  cells  present  into  epithelial,  erythrocytic,  and  leucocytic 
casts.  Epithelial  casts  contain  the  mononuclear  cells  of  the 
shed  renal  epithelium.  The  epithelial  cells  may  be  more  or 
less  degenerated,  and  their  nuclei  may  be  absent.  Some  cells 
may  appear  vacuolated,  others  granular.  Some  may  contain 
globules  of  fat.  The  cast  may  be  entirely  filled  with  cells,  or 
may  be  partly  hyaline.  The  presence  in  casts  of  epithelial 
cells,  not  greatly  degenerated,  is  practically  confined  to  the 
acute  stage  of  acute  nephritis.  Erythrocytic  casts  are  readily 
identified,  and  may  consist  of  casts  packed  with  obvious  and 
undegenerated  red  cells.  Some  casts  may  contain  both  red 
cells  and  epithelial  cells,  and  the  red  cells  may  be  more  or  less 
broken  up.  These  casts  are  similarly  found  in  acute  nephritis, 
and  particularly  in  the  acute  stages.  Free  red  cells  are 
always  present  in  the  urine  in  addition.  Leucocytic  casts, 
that  is  casts  which  contain  polynuclear  neutrophils,  are  not 
commonly  met  with.  They  may  be  found  in  septic  conditions 
of  the  kidney,  such  as  "  surgical "  or  "  consecutive  "  nephritis. 
A  few  polynuclear  cells  are  nearly  always  present  in  addition 
to  the  casts  in  a  case  of  acute  nephritis,  and  a  few  degenerated 
epithelial  cells  are  also  seen  lying  free. 

Granular  casts  are  casts  which  contain  no  formed 
elements  other  than  fine,  or  more  rarely  coarse,  granules. 
Granular  casts  are  perhaps  the  most  common  variety  seen, 
and  usually  predominate  in  chronic  parenchymatous  nephritis 
as  well  as  in  acute  nephritis  after  the  first  few  days  of  the 
disease.  The  majority  of  granular  casts  are  probably  derived 
in   the  same  way  as  the  epithelial  casts,  and  differ  only  in 


URINARY  DEPOSITS— URINARY   CALCULI.      255 

representing  a  later  stage  in  the  cells  which  have  now  undergone 
complete  granular  degeneration.  Granular  casts  may  less 
frequently  arise  from  a  granular  change  in  the  amorphous 
forms. 

Amorphous  casts. — The  commonest  variety  of  amorphous 
cast  is  the  hyaline  cast.  This  varies  considerably  in  size, 
and  may  be  very  large  and  spirally  twisted  in  its  long  axis. 
The  content  of  the  cast  is  as  a  rule  quite  structureless,  and 
almost  entirely  transparent.  These  casts  may  escape  observa- 
tion unless  the  light  reflected  through  the  preparation  is 
reduced  to  a  minimum.  Hyaline  casts  may  be  found  in  every 
form  of  nephritis,  and  may  be  the  only  variety  present  in  cases 
of  contracted  granular  kidney.  Very  occasionally  a  cast  of  this 
nature  may  be  found  in  the  urine  of  an  apparently  healthy 
person. 

Amyloid  casts  are  rarely  found,  and  are  more  refractile 
than  hyaline  casts.  They  are  frequently  fissured,  and  some- 
times give  the  amyloid  reaction  with  one  of  the  appropriate 
staining  mixtures  such  as  methyl  violet,  or  iodine  and  sulphuric 
acid.  Amyloid  casts  are  not  particularly  associated  with 
lardaceous  disease  of  the  kidneys. 

Spermatozoa  (Plate  XII.)  are  occasionally  found  in  the 
urine,  and  should  be  readily  recognised  by  their  shape.  The 
long,  thin  and  tapering  tail,  with  the  prominent  oval  head,  are 
quite  unmistakable.  Spermatozoa  in  the  urine  may  show 
active  movement  even  in  a  specimen  which  has  been  standing 
for  some  time.  Spermatozoa  are  readily  stained  by  the 
ordinary  dyes,  such  as  carbol-thionin. 

Animal  parasites  are  not  commonly  met  with  in  the  urine 
of  patients  in  this  country.  Occasionally  the  common  thread 
worm  (oxyuris  vermicularis)  or  its  ova  may  be  found  in  the  urine 
of  little  girls.  Filariee  are  very  rarely  detected  in  the  urine, 
even  in  those  countries  in  which  filariasis  is  common.  The 
only  parasitic  infection  of  the  urinary  tract  at  all  commonly 
met  with  is  that  of  bilharzia  haematobia.  In  cases  of 
hsematuria  coming  from  tropical  or  sub-tropical  climates,  such 
as  Egypt  or  parts  of  South  Africa,  where  this  parasite  is 
numerous,  its  presence  should  always  be  suspected.  Infection 
of  the  bladder  by  bilharzia  is  recognised  with  certainty  by  the 
detection  of  the  characteristic  ova  in  the  urine.  When  haenia- 
turia  is  present  the  ova  are  generally  numerous,  and  they  may 


256  CLINICAL   PATHOLOGY. 

persist  for  many  months  after  residence  in  an  infected  district. 
A  specimen  of  the  urinary  deposit  should  be  put  up  in  the 
ordinary  way,  and  the  ova  can  readily  be  seen  with  the  low 
power  of  the  microscope,  and  the  details  of  the  contained 
embryo  can  often  be  made  out  on  using  the  ^-inch  objective. 
If  the  ova  are  very  scanty  the  patient  should  be  told  to  empty 
his  bladder,  and  then  to  pass  the  last  few  drops  of  urine, 
evacuated  by  active  straining,  into  another  receptacle.  The 
ova  are  large  oval  bodies  with  a  definite  capsule  terminating 
in  a  single  sharp,  short  spine  which  is  quite  characteristic. 
Within  the  ovum  can  often  be  seen  a  coiled,  ciliated  embryo 
which  may  show  active  movement.  If  fresh  water  is  added  to 
the  urine  the  embryo  may  burst  its  way  out  of  the  ovum  and 
swim  about  freely.  The  ova  are  occasionally  found  in  the 
faeces,  and  in  this  situation  are  usually  provided  with  lateral 
and  not  terminal  spines. 

Unorganised  Urinary  Deposits. 

While  the  greater  number  of  the  organised  urinary  deposits 
are  only  present  in  the  urine  as  the  result  of  disease,  the 
majority  of  the  unorganised  deposits  are  not  necessarily  of 
any  pathological  import.  The  detection  of  a  crystalline  or 
non-crystalline  substance  in  the  urine  is  no  evidence  that  it  is 
being  excreted  in  excess.  The  only  conclusion  that  can  be 
drawn  is,  that  the  sample  of  urine  on  cooling  and  standing  is 
unable  to  contain  the  substance  in  solution.  The  majority  of 
normal  urines  and  almost  all  concentrated  urines  form  some 
deposit  on  standing.  Those  urines  from  which  no  deposit  can 
be  obtained,  even  after  standing  and  centrifuging,  are  nearly 
always  derived  from  cases  of  polyuria.  The  detection  of  uric 
acid  or  calcium  oxalate  crystals  in  the  urine  of  a  patient 
with  renal  symptoms  is  very  little  evidence  that  a  calculus 
of  similar  composition  will  be  found  in  the  kidney. 

The  following  are  the  more  important  unorganised 
deposits  : — 

Amorphous  urates  (Plate  XIII.). — The  deposition  of  amor- 
phous urates  from  an  acid  urine  on  cooling  is  an  extremely 
common  phenomenon.  The  urates  have  a  marked  tendency 
to  absorb  the  urinary  pigments,  and  a  concentrated  acid 
urine  will  frequently  throw  down  several  inches  of  a  thick 
pink  deposit  of  amorphous  urates.      Such  deposits  may  be 


URINARY  DEPOSITS— URINARY  CALCULI.      257 

found  in  almost  any  febrile  urine,  or  in  the  urine  of  a  perfectly 
normal  individual  who  has  recently  taken  an  unusual  amount 
of  exercise.  The  amorphous  urates  are  recognised  by  their 
occurrence  in  an  acid,  or  less  commonly,  in  a  neutral  urine,  by 
their  pinkish  colour,  by  the  manner  in  which  they  re-dissolve 
on  warming  the  urine,  and  by  their  appearance  under  the  micro- 
scope. Examined  under  the  microscope,  these  urates  appear 
as  fine,  loose,  amorphous  granules  of  a  yellowish-brown  tint. 
Almost  the  only  substance  they  can  be  mistaken  for  is  amor- 
phous phosphate.  The  phosphates  usually  form  coarser 
granules,  which  are  colourless,  which  dissolve  on  the  addition 
of  an  acid  but  not  on  warming,  and  which  usually  occur  in 
an  alkaline  urine.  The  amorphous  urates  consist  mainly  of 
acid  sodium  urate. 

Acid  ammonium  urate  (Plate  XIII.)  is  much  less 
commonly  met  with,  and  practically  only  occurs  in  alkaline 
urine.  It  may  be  found  in  normal  urine  which  has  been 
standing  for  a  long  time,  and  which  has  undergone  ammo- 
niacal  fermentation.  The  customary  crystalline  form  of 
ammonium  urate  is  a  very  striking  one.  The  crystals  are 
deeply  pigmented,  of  a  considerable  size,  and  of  very  irregular 
shape.  One  or  more  elongated  spinous  processes  proceed  from 
the  crystals,  giving  them  the  appearance  of  a  radish  with  its 
roots. 

Uric  acid  (Plate  XL).  —  Well-formed  crystals  of  uric 
acid  are  found  only  in  urines  of  acid  reaction.  They  occur 
under  much  the  same  conditions  as  do  deposits  of  amorphous 
urates.  Deposits  of  uric  acid  in  concentrated  urines  are  con- 
sequently of  no  significance,  but  a  continued  excretion  of 
urine  rich  in  such  deposits  is  associated  with  certain  patho- 
logical states  of  which  leukaemia  and  acute  gout  are  the  most 
noteworthy.  Uric  acid  crystallises  in  a  variety  of  forms 
which  differ  widely  in  size  and  shape.  Nearly  all  forms  are 
coloured  by  the  urinary  pigments,  but  colourless  uric  acid 
crystals  occur.  A  common  variety  of  uric  acid  deposit  is 
readily  visible  to  the  naked  eye  as  bright  red  crystals,  which 
have  a  tendency  to  adhere  to  the  sides  of  the  specimen  glass. 
These  crystals,  if  passed  in  considerable  amount,  are  referred 
to  by  the  patient  as  "gravel."  Under  the  microscope  the 
commonest  shape  of  the  uric  acid  crystal  is  that  of  the 
"  whetstone,"  and  a  number  of  such  crystals  united  together 

p.  17 


258  CLINICAL   PATHOLOGY. 

at  one  end  and  radiating  from  each  other  at  their  free  ends, 
constitute  the  macroscopic  gritty  red  granule.  Other  forms  of 
uric  acid  which  may  occur  are  crystals  somewhat  similar  to 
those  described  as  "  whetstones,"  but  flatter,  more  pointed,  and 
"  lozenge  "-shaped;  also  smaller  crystals,  more  quadrilateral 
in  shape  and  with  a  tendency  to  cling  together  in  clusters. 
Another  variety  of  crystal,  which  may  be  colourless,  is  almost 
circular,  and  presents  more  or  less  regular  striations  radiating 
from  the  centre  to  the  periphery.  Cones,  needles,  and  rod- 
like forms  are  less  commonly  met  with,  and  various  of  these 
abnormal  forms  are  doubtless  impure. 

Amorphous  phosphates  are  found  as  a  deposit  in  alkaline 
or  less  frequently  in  neutral  urine.  The  colourless  deposit  at 
the  bottom  of  a  test  tube  is  frequently  mistaken  for  pus,  and 
cannot  certainly  be  distinguished  from  it  by  naked-eye 
examination.  Under  the  microscope  the  deposit  is  seen  to 
consist  of  white  amorphous  granules,  not  unlike  those  of 
urates.  The  distinction  between  phosphates  and  urates  is 
given  under  the  description  of  the  latter.  The  amorphous 
phosphates  consist  of  calcium  and  magnesium  phosphate. 

Triple  phosphate  (Plate  XIIL),  or  ammonium  magnesium 
phosphate,  forms  the  crystalline  deposit  which  is  the  one 
most  commonly  met  with  in  ammoniacal  urine,  and  very 
rarely  in  slightly  acid  urine.  Deposits  containing  triple  phos- 
phates are  frequently  mixed  with  amorphous  phosphates, 
and  less  commonly  with  acid  ammonium  urate.  Triple 
phosphate  crystals  are  of  no  significance  in  a  stale  specimen 
of  urine,  and  in  comparatively  fresh  specimens  they  have  the 
same  meaning  as  the  alkaline  reaction  with  which  they  are 
associated.  Consequently  the  majority  of  freshly-voided 
alkaline  urines  which  contain  triple  phosphate  crystals  con- 
tain also  pus  and  micro-organisms.  The  crystals  are  usually  of 
considerable  size  and  of  somewhat  variable  shape.  In  almost 
all  samples  some  of  the  more  characteristic  forms  will  be 
present.  The  common  form  is  that  known  as  the  "  coffin  lid  " 
crystal,  and  is  a  straight  prism  with  obliquely-cut  terminal 
facets.     Less  common  are  feathery  and  fern-shaped  crystals. 

Stellar  phosphates  (Plate  XIII.)  or  crystals  of  calcium 
hydrogen  phosphate  maybe  found  in  neutral  or  nearly  neutral 
urine,  occasionally  in  health,  but  more  commonly  in  blood 
diseases  and  joint  affections.      These  crystals  are,  like  other 


PLATE   XIII. 


Triple  Phosphate  Crystals 


Calcium  Hydrogen  Phosphate  Crystals. 


o'gHj 

#»•            .                           ,  y_t 

•• "  'tf1*!*  'fpfitf 

Vw'. 

*  z^ 

&£«* 

'*:,:■<•'- 

- '"  >• 

Q 

O                □ 

<^\         o 

D 

0 

0 

D          O 

Amorphous  Urates 


Calcium  Oxalate  Crystals. 


Ammonium  Urate  Crystals. 


Cystine  Crystals. 


URINARY  DEPOSI TS— URINARY   CALCULI.      259 

phosphates,  readily  soluble  in  acetic  acid.  The  most  common 
form  assumed  by  the  crystals  is  that  of  a  long,  narrow  and 
flat  prism,  usually  pointed  at  one  end.  The  prisms  may  be 
collected  together  in  bunches  or  rosettes.  Less  commonly 
the  crystals  may  be  very  fine  and  feathery  and  collected  into 
tufts. 

Calcium  oxalate  (Plate  XIII.)  crystals  may  be  found  in  acid 
or  alkaline  urine,  but  more  frequently  in  the  former.  They 
often  occur  in  excess  after  the  ingestion  of  certain  fruits  and 
vegetables,  such  as  apples,  tomatoes  and  rhubarb.  It  is  stated 
that  a  continual  passage  of  calcium  oxalate  crystals  in  excess  is 
characteristic  of  chronic  pancreatitis.  The  crystals  are  com- 
monly found  in  perfectly  normal  urine,  particularly  if  it  has 
been  allowed  to  stand  for  some  hours,  and  they  may  form  a 
considerable  naked-eye  deposit  not  unlike  pus  in  appearance. 
The  type  of  crystal  most  commonly  found,  and  the  one  which 
accompanies  any  other  varieties  which  may  be  present,  is  a 
small  colourless  octahedron.  It  is  among  the  smallest  of  the 
urinary  crystals,  and  is  only  just  visible  under  the  §-inch 
objective,  the  ^-inch  being  required  for  identification.  When 
seen  from  above  with  the  pointed  end  uppermost  the  lines  of 
the  facets  show  as  intersecting  diagonals  on  a  square,  giving 
the  crystal  the  appearance  of  an  envelope.  In  addition  to  the 
"  envelope  "  forms,  oval,  spherical  and  dumb-bell  forms  are 
met  with.     The  crystals  are  practically  insoluble  in  acids. 

Calcium  carbonate,  commonly  found  in  the  urine  of 
herbivors,  is  rarely  met  with  in  human  urine.  It  may  occur 
in  alkaline  and  stale  urine,  and  usually  in  association  with 
phosphates.  The  deposit  may  be  crystalline,  the  ciystals 
having  the  shape  of  small  dumb-bells,  or  amorphous.  The 
nature  of  the  deposit  is  made  clear  by  the  addition  of  a  little 
dilute  hydrochloric  acid,  when  the  deposit  rapidly  dissolves 
with  effervescence. 

Cystine  (Plate  XIII.)  is  among  the  rarest  of  urinary  deposits, 
and  at  a  large  London  hospital  perhaps  one  case  of  cysti- 
nuria  will  come  under  observation  in  five  years.  Nevertheless 
the  student  is,  for  examination  purposes,  expected  to  be  very 
familiar  with  the  cystine  crystal.  The  condition  is  an  interest- 
ing one,  since  it  forms  one  of  the  rare  congenital  abnormali- 
ties of  metabolism,  and  its  occurrence  appears  to  be  confined 
to  the  children  of  first  cousins.     Although  traces  of  cystine 

17—2 


260  CLINICAL   PATHOLOGY. 

can  be  obtained  from  the  normal  urine,  the  occurrence  of 
cystine  crystals  in  the  urine  is  clear  evidence  of  this  rare 
condition.  Cystinuria  may  persist  for  years  without  producing 
any  symptoms ;  it  is,  however,  frequently  associated  with  the 
formation  of  renal  calculi  composed  of  cystine.  The  deposition 
of  the  crystals  takes  place  in  acid  or  faintly  alkaline  urine. 

The  appearance  of  the  crystals  under  the  microscope  is 
very  characteristic,  since  they  consist  of  flat,  colourless, 
regular,  hexagonal  plates.  All  the  crystals  in  any  one  deposit 
will  not  be  regular,  but  a  certain  number  of  perfectly  formed 
ones  are  nearly  always  found.  Cystine  is  readily  soluble  in 
ammonia,  but  is  insoluble  in  water,  ether,  or  acetic  acid. 

Leucine  and  tyrosine. — These  substances,  which  result 
from  the  decomposition  of  proteids,  are  not  found  in  normal 
urine.  The  conditions  in  which  they  are  most  frequently 
met  with  are  acute  yellow  atrophy  of  the  liver  and  phosphorus 
poisoning.  The  two  crystals  almost  always  occur  together 
in  the  urine,  and  they  may  form  a  considerable  naked-eye 
deposit  in  the  specimen  glass.  Tyrosine  is  less  soluble  than 
leucine,  and  separates  out  from  the  urine  more  readily  and  in 
greater  abundance. 

Tyrosine  (Plate  XII.)  crystals  take  the  form  of  brush-like 
tufts  of  very  fine  needles,  and  are  either  colourless  or  greenish- 
yellow. 

To  obtain  the  crystals  in  a  pure  form  from  the  urine : — 

Eemove  coagulable  proteids,  if  present,  by  boiling  and 
filtering. 

Precipitate  the  pigments  and  extractives  by  basic  lead 
acetate. 

Shake  well.     Allow  to  stand.     Filter. 

Pass  H2S  through  the  filtrate  to  remove  the  lead. 

Filter  and  evaporate  down  the  clear  filtrate. 

Colourless  crystals  of  tyrosine  separate  out  on  cooling. 

The  material  obtained  on  evaporation  can  be  further  tested 
for  tyrosine  by  Hofmann's  reaction.  In  this  reaction  the 
material  is  heated  with  water  in  a  test  tube,  and  a  few7  drops 
of  Millon's  reagent  are  added  to  the  hot  solution,  which 
becomes  rose-coloured  in  the  presence  of  tyrosine. 

Leucine  crystals  take  the  form  of  yellow  spheroids,  which 
show  both  radial  and  concentric  striation.  Leucine  is  readily 
soluble  in  acids  and  alkalies. 


URINARY  DEPOSITS— URINARY  CALCULI.      261 

Foreign  bodies  in  urinary  deposits.— A  complete  list 
of  the  adventitious  material  which  may  be  found  in  the  urine 
would  rival  the  well-known  list  of  foreign  bodies  met  with 
in  the  vagina,  though  there  is  no  record  of  a  bust  of 
Napoleon  in  a  specimen  glass.  Patients  may,  however, 
deposit  almost  any  available  object  in  the  urine.  The  follow- 
ing include  some  of  the  more  common  adventitious  objects 
which  may  be  puzzling  to  the  student. 

Marks  on  the  cover-glass  have  been  frequently  and 
earnestly  scrutinised  with  a  view  to  interpretation,  and  the 
mistake  is  easily  made.  If  the  cover-glass  is  not  scrupulously 
clean  and  it  is  difficult  to  make  it  so,  or  if  it  is  scratched, 
the  dust  and  scratches  on  its  surface  are  the  objects  first 
seen  on  focussing  the  objective  down.  The  scratches  may 
assume  fantastic  shapes  and  may  even  remotely  resemble 
casts,  while  the  surrounding  dust  is  mistaken  for  the  urinary 
debris.  The  mistake  is  at  once  rectified  by  continuing  to 
focus  down  until  the  actual  urinary  deposit  comes  into  view. 
Scratches  on  the  slide  itself  may  of  course  be  similarly  mis- 
taken for  urinary  contents,  and  have  to  be  differentiated  by 
their  shape  and  appearance.     They  present  no  real  difficulty. 

Air  bubbles  should  not  be  present  in  a  carefully  put  up 
sample  of  deposit ;  still  they  sometimes  occur.  They  are 
circular  in  form  unless  compressed.  They  are  recognised  by 
their  broad,  dark  and  often  double  outlines,  and  their  clear, 
shining  centres. 

Fat  globules  may  occur  in  disease,  but  are  more  frequently 
present  as  a  foreign  body  in  catheter  specimens.  They  are 
recognised  by  their  circular  form,  their  variability  in  size, 
their  sharp  margins  and  refractile  centres.  They  turn  black 
on  adding  osmic  acid. 

Starch  granules  are  fairly  frequently  found  in  the  urine, 
particularly  of  children,  their  source  being  the  dusting  powder 
used.  They  are  recognised  by  their  oval  shape  and  concentric 
lamellation.     They  turn  blue  on  the  addition  of  iodine. 

Sulphur  granules  are  occasionally  found  in  the  deposit 
of  urine  which  has  been  subjected  to  the  sulphur  test  for 
bile  salts.  The  granules  appear  as  dark  irregular  clumps  of 
crystalline  bodies. 

Hairs  are  commonly  present  in  the  specimen  glass,  and 
are  usually  pretty  obvious  to  the  naked  eye.     They  are  as 


262  CLINICAL   PATHOLOGY. 

a  rule  pigmented,  and  their  structure  becomes  more  obvious 
on  the  addition  of  caustic  soda. 

Fibres  of  cotton  or  linen  are  very  commonly  met  with,  and 
are  usually  derived  from  the  cloth  used  in  cleaning  the  speci- 
men glass.  They  are  necessarily  of  variable  size  and  shape,  but 
are  all  more  or  less  cylindrical  and  twisted,  and  usually  have 
frayed  ends.  The  only  pathological  objects  for  which  they  can 
be  mistaken  are  casts,  and  the  resemblance  is  not  particularly 
striking. 

Urinary  Calculi. 

The  calculi  which  may  be  found  in  an}-  part  of  the  urinary 
tract  can  be  divided  into  those  which  are  comparatively 
common  and  those  which  are  extremely  rare.  We  are  only 
concerned  here  with  the  composition  of  the  various  calculi,  and 
this  is  readily  determined  by  a  short  and  simple  scheme  of 
analysis.  The  exact  percentage  composition  of  mixed  calculi 
is  naturally  a  more  laborious  proceeding,  but  does  not  form  a 
part  of  ordinary  clinical  pathology. 

Speaking  generally,  calculi  may  vary  in  size  from  a  small 
concretion,  such  as  ma}7  be  passed  by  the  urethra,  to  a  mass 
the  size  of  a  clenched  fist.  They  are  conmionry  pigmented. 
They  may  be  smooth  or  rugged.  They  tend  to  take  the  shape 
of  the  viscus  in  which  they  have  been  formed,  as,  for  example, 
the  pelvis  of  the  kidney.  If  multiple  they  may  be  facetted. 
The  fractured  surface  frequently  shows  concentric  rings. 
Calculi  are  commonly  of  mixed  composition,  and  the  nucleus 
may  be  formed  of  a  different  material  to  the  cortical  portion. 
The  comparatively  common  stones  are  calcium  oxalate,  uric 
acid,  ammonium  urate  and  phosphatic  calculi. 

Calcium  oxalate  stones  mixed  with  a  certain  amount 
of  calcium  phosphate  would  appear  to  be  the  most  common 
variety.  Calcium  oxalate  calculi  are  as  a  rule  hard,  of  a  reddish- 
brown  colour,  and  with  a  granular  surface  which  has  given  them 
the  name  of  "  mulberry  "  calculi.     They  may  be  branched. 

Calculi  of  triple  phosphate  are  white  and  crumbling, 
are  practically  only  found  in  the  bladder,  and  may  be  formed 
around  a  foreign  bod}7. 

Uric  acid  calculi  are  less  common  and,  contrary  to  the 
statements  of  many  text -books,  are  extremely  rarely  found  in 
the  kidney,  though  the}7  are  not  very  infrequently  met  with  in 
the  bladder.     The  rarity  of  uric  acid  calculi  in  the  kidney  is 


URINARY  DEPOSITS— URINARY  CALCULI.      263 

fortunate,  since  the  permeability  of  uric  acid  to  the  X-rays 
is  the  same  as  that  of  the  belly- wall ;  consequently  they  can- 
not be  recognised  by  X-ray  examination.  The  calculi  are 
usually  small,  chocolate-coloured,  fairly  hard,  and  have  a 
smooth  surface. 

While  pure  uric  acid  calculi  are  rare,  uric  acid  is  fairly 
commonly  found  in  association  with  some  other  substance 
such  as  calcium  phosphate  or  oxalate.  The  mixed  calculus 
can  be  recognised  by  X-ray  photography. 

Urate  stones  consisting  of  ammonium,  with  a  lesser 
amount  of  sodium  urate,  are  hard  stones  of  a  brown  colour. 

Calcium  phosphate  is  not  a  common  variety  of  calculus, 
but  a  certain  amount  of  this  substance  is  not  infrequently 
present  in  association  with  calcium  oxalate,  triple  phosphate 
and,  occasionally,  calcium  carbonate. 

The  composition  of  the  commoner  calculi  should  be  sought 
according  to  the  following  scheme  of  examination.  If  the 
reactions  for  those  substances  are  not  given,  or  only  traces  of 
them  are  found,  some  of  the  rarer  substances  must  be  tested 
for,  as  subsequently  described. 

The  chemical  examination  of  urinary  calculi. 

If  the  stone  is  of  considerable  size,  cut  it  in  half. 

Examine  the  cut  surface,  and  if  the  nucleus  differs  in 
appearance  from  the  cortical  portion,  scrape  out  the  nucleus 
first  and  examine  it  separately. 

Powder  up  the  calculus  in  a  clean  mortar. 

Then  apply  each  of  the  following  tests  : — 

(1)  Place  a  little  powder  in  the  bottom  of  a  clean  test  tube 
and  add  dilute  HC1. 

If  effervescence  occurs  carbonates  are  present. 

If  effervescence  is  doubtful,  place  some  powder  on  a  slide 
with  a  cover -glass  over  it.  Run  the  dilute  acid  between  slide 
and  cover-glass  and  watch  under  the  microscope  for  the 
evolution  of  gas  bubbles  from  the  granules  of  powder. 

Carbonates  are  rarely  obtained  except  as  traces. 

(2)  Place  a  little  powder  on  a  platinum  foil. 
Heat  over  the  Bunsen  flame. 

When  the  powder  is  incinerated  shake  it  out  into  a  clean 
test  tube. 

Add  dilute  HC1  as  in  1. 


264  CLINICAL   PATHOLOGY. 

Oxalates  were  present  if  effervescence  occurs.  If  in  doubt 
repeat  on  a  glass  slide  under  the  microscope.  The  oxalates 
were  converted  into  carbonates  by  the  heating  on  the  platinum 
foil. 

If  oxalates  or  carbonates  are  present  the  base  may  be 
presumed  to  be  calcium. 

The  presence  of  calcium  may,  if  preferred,  be  confirmed  by 
the  usual  tests  in  solution,  for  example  : — 
+  Ammonium  carbonate  =  white  precipitate. 
+  Ammonium  oxalate      =  white    precipitate,  insoluble  in 

acetic  acid ;  soluble  in  dilute 
HC1. 
+  Calcium  sulphate  =  no  precipitate. 

(3)  Place  a  little  powder  in  a  clean  porcelain  crucible,  and 
perform  the  murexide  test  as  follows  : — 

Add  a  few  drops  of  nitric  acid. 

Heat  on  a  sand  bath  in  the  fume  cupboard. 

If  an  effervescence  of  gas  takes  place  and  a  bright  red 
residue  is  left: — 

Allow  to  cool. 

With  a  glass  rod  add  to  a  portion  of  the  residue  1  drop  of 
caustic  soda.     The  residue  turns  bluish-violet. 

To  another  portion  of  the  residue  add  1  drop  of  ammonia. 
The  residue  turns  purple. 

A  positive  murexide  test  is  evidence  of  the  presence  of  Uric 
Acid  or  Ammonium  Urate. 

To  determine  which  is  present : — 

Place  some  fresh  powder  in  a  test  tube. 

Add  a  little  caustic  soda  and  heat. 

Examine  for  the  evolution  of  ammonia  by  the  smell  and  by 
a  piece  of  moist  litmus  paper  held  in  the  mouth  of  the  test 
tube. 

If  ammonia  is  detected  the  material  contained  ammonium 
urate  ;  if,  as  is  more  usual,  no  ammonia  is  present,  the  material 
was  uric  acid. 

(4)  Dissolve  some  of  the  powder  in  dilute  warm  HC1  in  a 
test  tube. 

If  necessary  filter. 

Add  a  few  drops  of  nitric  acid. 


URINARY  DEPOSITS- URINARY  CALCULI,      265 

Add  ammonium  molybdate  solution  in  quantity  equal  to  the 
amount  of  the  original  solution.  The  ammonium  molybdate 
solution  must  be  comparatively  fresh,  and  there  should  be  little 
or  no  precipitate  in  the  bottom  of  the  bottle. 

Heat  and  allow  to  stand. 

If  a  yellow  precipitate  forms,  phosphates  are  present. 

The  phosphates  may  be  earthy  phosphates  or,  less  frequently, 
ammonio-magnesium  phosphate. 

To  distinguish  between  the  phosphates  test  for  the  presence 
of  ammonia  as  in  (3). 

If  any  of  the  above  tests  are  strongly  positive  it  is  not 
necessary  to  proceed  further.  But  if  the  tests  are  negative  or 
only  traces  of  the  above  substances  are  found,  the  following 
rare  constituents  of  urinary  calculi  must  be  sought  for  : — 

Cystine. — Burn  the  powder  on  a  platinum  foil  over  the 
flame. 

Cystine  burns  rapidly  with  a  blue  flame  and  gives  off  a 
sharp  odour. 

Dissolve  some  powder  in  ammonia. 

Cystine  is  readily  soluble,  and  on  spontaneous  evaporation 
of  the  ammonia  the  typical  hexagonal  plates  separate  out. 

Xanthine. — The  powder  on  the  platinum  foil  burns  away, 
but  without  a  flame. 

Try  the  murexide  test.  There  is  no  effervescence  on  adding 
nitric  acid,  and  the  residue  left  after  heating  is  yellow. 

Add  a  drop  of  caustic  soda  to  the  dried  residue,  after  cooling ; 
in  the  presence  of  xanthine  the  residue  becomes  orange. 
Warm,  and  the  colour  changes  to  red. 

Fibrin. — The  powder  burns  on  the  platinum  foil  with  a 
yellow  flame  and  an  odour  of  burnt  feathers.  The  powder  is 
insoluble  in  alcohol  or  ether,  but  soluble  in  hot  caustic  potash. 

Add  acetic  acid  to  the  caustic  potash  solution.  The  fibrin 
is  precijDitated  with  an  evolution  of  H2S. 

Urostealith. — The  powder  burns  with  a  yellow  flame  and 
an  odour  of  resin. 

It  is  soluble  in  alcohol  and  ether. 


CHAPTER    XIX. 

special  investigations  of  the  trine — bacteriology   of  the 
urino- genital  tract. 

Special  Investigations  of  the  Urine. 

By  special  investigations  are  meant  those  which  are  outside 
the  ordinary  routine  examination  of  the  urine  and  those  which 
are  not  commonly  called  for  even  if  any  abnormal  substance 
is  detected.  There  exist  a  very  large  number  of  such  investi- 
gations, but  the  majority  of  them  are  of  more  scientific  than 
practical  interest  and  cannot  be  described  here.  Only  those 
methods  are  given  which  have  some  bearing  upon  the  clinical 
diagnosis  or  treatment  of  disease,  and  which  do  not  involve 
any  very  specialised  knowledge  of  chemistry. 

The  estimation  of  uric  acid. — Uric  acid  belongs  to  the 
group  of  bodies  known  as  purines,  and  is  the  chief  nitrogenous 
excretory  product  of  birds.  In  man  the  average  daily  output 
of  uric  acid  varies  from  0*5  to  1  gramme.  The  amount  is 
increased  with  any  abnormal  destruction  of  nuclei,  as  in  the 
leukaemias,  when  four  or  five  times  the  normal  quantity  may  be 
excreted.  A  lesser  increase  accompanies  high  fever.  In  cases 
of  gout  the  uric  acid  output  is  markedly  low  in  the  intervals 
of  freedom,  but  just  before  an  attack  and  during  the  height 
of  the  attack  a  marked  increase  in  the  output  takes  place. 
Hopkins'  method  of  estimating  the  uric  acid  in  the  urine  as 
modified  by  Folin  is  that  most  commonly  employed. 

The  following  solutions  are  required  : — 

N 

(1)  A  standard   ^   solution  of    potassium  permanganate. 

1  c.c.  of  this  solution  =  0*00375  gramme  uric  acid. 

(2)  A  10  per  cent,  solution  of  ammonium  sulphate. 

(3)  A  solution  containing — 

Uranium  acetate       ....  5  grammes. 

Ammonium  sulphate          .         ,         .  500       ,, 

10  per  cent,  acetic  acid      ...  60  c.c. 

Water 650   „ 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  267 

(4)  A  measured  24  hours'  supply  of  urine,  which  must  be 
well  stirred  before  a  sample  is  taken. 

Proceed  as  follows  : — 

Stage  1. — Into  a  500  c.c.  flask  measure  200  c.c.  of  urine 
and  50  c.c.  of  the  uranium  acetate  solution. 

Mix  and  allow  to  stand  for  half  an  hour,  or  until  the 
precipitate  has  settled. 

By  this  procedure  a  mucoid  substance  is  precipitated  from 
the  urine,  which  would,  if  left,  render  subsequent  filtration  of 
the  ammonium  urate  very  slow  and  tedious. 

Stage  2. — Filter  the  bulk  of  the  supernatant  fluid  through 
a  dry  filter  paper  into  a  dry  vessel. 

Measure  125  c.c.  of  this  (containing  100  c.c.  of  the  original 
urine)  into  a  beaker. 

Add  5  c.c.  of  concentrated  ammonia. 

Mix  and  allow  to  stand  for  from  12  to  24  hours. 

In  this  stage  the  uric  acid  is  precipitated  as  ammonium 
urate,  which  is  insoluble  in  the  presence  of  the  ammonium 
sulphate. 

Stage  3.— Filter. 

Wash  the  precipitate  on  to  the  filter  with  the  10  per  cent, 
ammonium  sulphate  solution. 

Wash  two  or  three  times  with  the  same  solution  to  remove 
the  chlorides.  Remove  the  filter  paper.  Open  it.  WTash  off 
the  precipitate  into  a  beaker  with  a  stream  of  hot  distilled 
water. 

Stage  4. — The  ammonium  urate  precipitate  is  now 
suspended  in  about  100  c.c.  of  distilled  water. 

Add  15  c.c.  of  concentrated  sulphuric  acid. 

Titrate  with  the  permanganate  solution  while  warm. 

The  titration  is  completed  when  a  faint  permanent  pink 
colour  is  diffused  through  the  solution. 

In  performing  the  titration  a  burette  with  a  glass  tap  must 
be  used,  since  the  permanganate  acts  upon  rubber. 

The  calculation  is  made  from  the  number  of  cubic  centimetres 
of  the  permanganate  solution  used,  and  this  number  multiplied 
by  0*00375  gives  the  number  of  grammes  of  uric  acid  in  100  c.c. 
of  urine.  On  account  of  the  solubility  of  ammonium  urate, 
however,  a  correction  has  to  be  made,  and  0'003  gramme 
of  uric  acid  should  be  added  for  every  100  c.c.  of  urine 
used. 


268  CLINICAL   PATHOLOGY. 

The  estimation  of  purines. — The  purine  bases  include 
purine,  xanthine,  and  hypoxanthine,  and  are  increased  in  the 
leukaemias  and  in  febrile  conditions.  The  "purine  bodies" 
include  the  purine  bases  and  uric  acid,  and  they  can  be  readily 
estimated  by  the  "Walker  Hall  purinometer,  -with  sufficient 
accuracy  for  clinical  purposes.  The  variations  in  such  a 
disease  as  myeloid  leukaemia  are  very  considerable. 

The  purinometer  is  provided  with  full  instructions  for  use, 
which  may  be  recapitulated  here. 

The  apparatus  consists  of  a  tall  glass  vessel  divided  by  a  stop- 
cock into  a  lower  reservoir  and  an  upper  graduated  cylinder. 

The  following  solutions  are  required  : — 

(1)  Magnesia  mixture     ....       100  c.c. 
20  per  cent,  ammonia  solution  .         .       100    ,, 
Pure  talc  (finely  ground)  ...       10  grammes. 

The  magnesia  mixture  has  the  following  composition  : — 
Magnesium  chloride  crystals    .         .       100  grammes. 
Ammonium  chloride 
Ammonia         .... 
Water  to  .... 

(2)  Silver  nitrate    .... 
Ammonia  (strong)     . 

Talc 

Distilled  water 

The  solutions  may  be  made  in  bulk  and  kept. 

(3)  A  measured  21  hours  sample  of  urine. 

To  use  the  instrument  close  the  stopcock  and  pour  in  urine 
(previously  freed  from  albumin  if  necessaiy)  to  the  90  c.c. 
mark. 

Open  the  stopcock. 

Add  20  c.c.  of  the  No.  1  solution. 

Shake  well  and  allow  to  stand  until  all  the  precipitate  of 
phosphates  has  settled  into  the  lower  chamber. 

Close  the  stopcock. 

Add  No.  2  solution  until  the  total  fluid  reaches  the  100  c.c. 
mark. 

Continue  to  rotate  the  cylinder  until  the  precipitate  in  it 
has  become  a  yellowish  white. 

Stand  in  a  room  of  even  temperature  away  from  the  light 
for  21  hours. 

Read  the  upper  level  of  the  precipitate  in  the  cylinder. 


110 
250 
1,000  c.c. 

1  gramme. 
100  c.c. 

5  grammes. 
100  c.c. 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  269 

A  scale  is  provided  which  gives  the  percentage  of  purine 
bodies  in  the  urine  corresponding  to  the  number  of  cubic 
centimetres  of  precipitate. 

The  estimation  of  creatine.— Creatinine  is  a  normal 
constituent  of  the  urine,  and  about  1  gramme  of  it  is  excreted 
daily.  It  is  the  anhydride  of  creatine  and  is  obtained 
from  it  by  boiling  with  an  acid.  Creatine  is  not  found  in  the 
urine  under  normal  circumstances.  Creatinine  is  supposed  to 
be  derived  from  either  ingested  or  muscle  creatine ;  but  modern 
experimental  physiology  is  partly  opposed  to  this  view,  for 
the  daily  output  of  creatinine  is  very  constant  whatever  the  diet 
of  the  individual.  Excess  of  creatinine  in  the  food  is  excreted 
as  creatinine,  and  excess  of  creatine  as  creatine. 

The  seat  of  formation  of  creatinine  is  held  by  some  to  be  the 
liver,  and  in  certain  diseases  of  the  liver  creatine  is  found  in 
considerable  amount  in  the  urine.  It  is  stated  that  a  neoplasm 
of  the  liver,  whether  primary  or  secondary  in  origin  and 
however  small,  leads  to  an  excretion  of  creatine.  A  similar 
excretion  takes  place  with  a  liver  abscess,  but  not  with 
cirrhosis  of  the  liver.  Creatine  may  also  appear  in  the  urine 
in  grave  wasting  diseases,  or  in  any  condition  associated  with 
acidosis.  Should  these  statements  be  confirmed,  and  there  is 
evidence  in  their  support,  the  detection  and  estimation  of 
creatine  in  the  urine  should  in  certain  cases  be  a  valuable  aid 
to  clinical  diagnosis.  In  the  absence  of  severe  wasting  a 
positive  creatine  estimation  would  be  strongly  in  support  of 
the  diagnosis  of  hepatic  neoplasm  in  doubtful  cases  of  jaundice. 
The  negative  test  is  more  reliable,  since  absence  of  creatine 
from  the  urine  in  cases  of  gastric  or  other  abdominal  carcino- 
mata  would  be  in  favour  of  operative  treatment  in  so  far  as 
indicating  that  dissemination  of  the  growth  in  the  liver  had 
not  yet  taken  place. 

The  actual  estimation  of  creatine  is  comparatively  rapid 
and  simple,  but  requires  a  special  apparatus.  The  estimation 
is  performed  in  two  parts.  In  the  first  part  the  creatinine  is 
estimated.  In  the  second  the  creatine  which  may  be  present 
is  converted  by  hydrolysis  with  an  acid  into  creatinine,  and 
the  total  creatinine  again  estimated.  The  difference  between 
the  two  estimations  represents  the  creatine. 

The  apparatus  required  for  the  estimation  of  creatinine  is  a 


270  CLINICAL   PATHOLOGY. 

somewhat  expensive  one,  and  is  known  as  the  Duboscq  colori- 
meter.    The  colour  estimation  depends  upon  the  fact  that  a 

layer  of  -~   potassium  bichromate  8  mm.   deep  has  the  same 

colour  as  a  layer  of  a  solution  made  from  10  rngrni.  of 
creatinine,  picric  acid  and  caustic  soda  8*1  mm.  deep.      By  a 

comparison  of  a  solution  of  jr  potassium  bichromate  with  a 

solution  of  picric  acid  and  caustic  soda  containing  an  unknown 
quantity  of  creatinine  the  amount  of  creatinine  present  can 
be  reckoned  from  the  depth  of  the  solution,  which  corresponds 
in  colour  with  the  bichromate  solution  8  mm.  deep. 

The  estimation  of  creatine  is  performed  as  follows : — 

A  sample  from  a  "2±  hours  specimen  of  urine  should  be 
used. 

Part  1. — To  estimate  the  preformed  creatinine. 

To  10  c.c.  of  urine  in  a  500  c.c.  measure  add  15  c.c.  of  a 
saturated  aqueous  solution  of  picric  acid  and  5  c.c.  of  10  per 
cent,  caustic  soda.     Shake  well. 

Stand  5  minutes  at  room  temperature. 

Make  up  to  500  c.c.  with  distilled  water  and  mix. 

In  one  cylinder  of  the  Duboscq  colorimeter  place  «-  potassium 

bichromate  solution  and  set  the  glass  plunger  at  8  mm. 
depth. 

Place  the  treated  urine  in  the  other  cylinder  and  by  means 
of  the  screw  move  the  plunger  up  and  down  until  the  colour 
of  the  two  solutions  is  the  same. 

Use  davlight  and  take  the  mean  of  at  least  6  readings. 

The  calculation  is  reliable  at  readings  between  depths  of 
5  and  12  mm. 

The  calculation  is  performed  as  follows  : — 

When  8'1  mm.  treated  urine  =  8  mm.  of  the  standard 
solution,  then  10  rngrni.  of  creatinine  were  present  in  the 
10  c.c.  of  urine  taken. 

Suppose  the  average  reading  to  be  6  mm.,  then  10  c.c.  of 

„         •  ,  .      10X8-1  , 

the  untie  contains ^ rngrni.  ot  creatinine. 

Part  2. — To  convert  creatine  into  creatinine. 
Take  10  c.c.  of  urine  in  a  small  flask  and  add  5  c.c.  of 
3*5  per  cent.  HC1. 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  271 

Heat  at  120°  C.  in  the  autoclave  for  30  minutes. 

Neutralise  the  HC1.  exactly  with  strong  caustic  soda. 

Cool  to  room  temperature;  add  15  c.c.  of  the  picric  acid 
solution  and  5  c.c.  of  the  10  per  cent,  caustic  soda. 

Stand  5  minutes  and  wash  out  with  distilled  water  up  to 
500  c.c.  in  a  large  measure  glass. 

Repeat  the  colour  estimation  as  in  Part  1. 

Calculation : 

/NH*  Minus  /NH\, 

Creatine  =  NH-CV  w  f      =  Creatinine  =  NH-C 

\N-CHoCOOH      vvatei  \XT  I 

,  *  x  N  -  CH2 

CH*  ok 

(Molecular  weight  =  131)  (Molecular  weight  =  113) 

m  =  M6. 

113 

Suppose  reading  of  preformed  creatinine  =11 
and  of  total  creatinine  .         .         .         =8 


Then  preformed  creatinine 
and  total  creatinine 


10  X  8-1 

11 
10  X  8-1 


The  difference  multiplied  by  116  =  creatine  present  in 
10  c.c.  of  urine. 

Estimation  of  phosphates.  A  method  of  estimating  the 
phosphates  in  urine  finds  a  place  in  the  majority  of  text-books 
and  is  given  here.  The  estimation  cannot  be  said  to  have 
any  valuable  bearing  at  present  upon  ordinary  clinical 
medicine,  and  in  the  majority  of  cases  the  elimination  of 
phosphates  by  the  urine  varies  directly  with  that  of  uric 
acid. 

The  phosphates  can  be  estimated  by  the  following  volu- 
metric method,  which  depends  upon  the  precipitation  of  the 
phosphates  by  uranium  nitrate  in  the  presence  of  acid  sodium 
acetate.  The  indicator  used  is  tincture  of  cochineal,  which 
becomes  green  in  the  presence  of  excess  of  uranium  nitrate. 

A  standard  uranium  nitrate  solution  is  used,  1  c.c.  of 
which  =0  005  gramme  P205. 

The  acid  sodium  acetate  solution  is  made  by  dissolving 
100  grammes  sodium  acetate  in  a  little  water,  adding  100  c.c. 
of  strong  acetic  acid,  and  making  up  to  1  litre  with  water. 


272  CLINICAL   PATHOLOGY. 

The  estimation  is  performed  as  follows : — 

Measure  50  c.c.  of  urine  into  a  beaker. 

Add  5  c.c.  of  the  sodium  acetate  solution. 

Add  a  few  drops  of  cochineal  tincture. 

Heat  the  urine  to  boiling,  and  run  in  the  standard  uranium 
nitrate  solution  so  long  as  a  precipitate  is  formed. 

Continue  to  run  the  uranium  solution  into  the  boiling  urine 
drop  by  drop  until  the  red  colour  becomes  green. 

To  calculate  the  result : — 

Multiply  the  number  of  cubic  centimetres  of  uranium 
solution  used  by  0*005,  and  the  result  is  the  number  of 
grammes  of  P205  in  50  c.c.  of  urine. 

The  average  daily  excretion  of  P205  is  from  2  to  3  grammes. 

Estimation  of  sulphates. — The  sulphates  of  the  urine  are 
of  two  kinds — the  inorganic,  combined  with  sodium  and 
potassium,  and  the  organic  or  ethereal,  combined  with  cresol, 
phenol,  indole,  scatole,  etc.  The  inorganic  sulphates  are  greatly 
in  excess  of  the  ethereal.  The  total  sulphates  are  increased 
in  febrile  conditions  and  the  ethereal  sulphates  with  stasis  of 
the  intestinal  contents  and  in  suppurative  processes. 

The  most  reliable  methods  of  estimating  the  sulphates  are 
gravimetric  and  are  too  long  to  be  described  here.  The  estima- 
tion of  the  total  sulphates  only  has  little  significance. 

Estimation  of  chlorides. — The  chlorides  in  the  urine 
consist  almost  entirely  of  sodium  chloride,  and  necessarily 
vary  with  the  amount  of  salt  ingested.  The  chlorides  are 
diminished  in  all  febrile  conditions,  and  are  very  markedly 
diminished  in  lobar  pneumonia.  The  diminution  of  the 
chlorides  of  the  urine  in  pneumonia  is  more  extreme  than  in 
almost  any  other  condition,  and  is  of  diagnostic  significance  ; 
the  return  of  the  chlorides  to  normal  or  above  the  normal  in 
this  disease  commences  after  the  crisis. 

The  amount  of  chloride  in  the  urine  can  be  roughly  tested 
by  filtering  some  urine  into  a  test  tube,  adding  a  few  drops 
of  pure  nitric  acid  and  then  silver  nitrate  solution  until  no 
more  precipitate  occurs.  A  thick,  white,  curdy  precipitate  of 
chlorides  takes  place  in  normal  urine-  In  cases  of  pneumonia, 
only  a  faint  cloud  forms. 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  273 

The  chlorides  can  be  estimated  by  the  Volhard  process. 

The  principle  of  the  method  consists  in  adding  to  the  urine 
an  excess  of  a  standard  solution  of  silver  nitrate  and  then 
estimating  this  excess  by  titrating  the  mixture  with  a  standard 
solution  of  potassium  sulpho-cyanide,  iron  alum  being  used 
as  the  indicator. 

The  estimation  is  performed  as  follows  : — 

Measure  10  c.c.  of  urine  into  a  100  c.c.  measure. 

Add  about  4  c.c.  of  pure  nitric  acid. 

N 
Add  20  c.c.  of  —  silver  nitrate  solution. 

Make  up  to  100  c.c.  with  distilled  water. 
Mix  thoroughly   and   filter    50   c.c.   of   the  mixture  into  a 
beaker. 

Add  about  5  c.c.  of  a  freshly-prepared  solution  of  iron  alum. 

Run  in  —  potassium  sulpho-cyanide  solution  until  a  reddish- 
brown  colour  which  remains  for  3  minutes  is  obtained. 

To  calculate  the  result  :  — 

The  number  of  cubic  centimetres  of  ammonium  sulpho- 
cyanide  used  corresponds  to  the  number  of  cubic  centimetres 
of  silver  nitrate  in  excess,  and  has  to  be  deducted  from  the 
10  c.c.  added  to  the  5  c.c  of  urine  (only  half  the  quantity  of 
the  mixture  of  urine  and  silver  solution  was  titrated).  The 
difference  in  cubic  centimetres  represents  the  chlorides  in 
5  c.c.  of  urine,  and  this  difference  multiplied  by  '00585  = 
the  grammes  of  chlorides,  reckoned  as  NaCl,  in  5  c.c.  of  urine. 
To  obtain  the  percentage  multiply  by  20. 

The  average  daily  excretion  of  chlorides  is  about  12 
grammes. 

Cammidge's  pancreatic  reaction.— The  position  which 
this  reaction  holds  among  chemical  aids  to  clinical  diagnosis 
is  a  matter  of  considerable  dispute.  The  reaction  has  been 
so  widely  used  that  it  is  desirable  to  describe  it  here. 

Cammidge's  original  reaction  was  a  double  one,  consisting 
of  two  separate  analyses  described  as  test  A  and  test  B.  From 
the  nature  of  the  crystals  found  in  these  two  tests  it  was 
claimed  that  in  the  majority  of  instances  not  only  could  the 
existence  of  a  pancreatic  lesion  be  recognised,  but  also  the 
nature   of  the   lesion   present.      The  original  theory   of   the 

18 


274  CLINICAL   PATHOLOGY. 

reaction  appears  to  have  been  broadly  as  follows.  An  escape 
of  the  pancreatic  juice  leads  to  fat  necrosis,  with  the  splitting 
of  neutral  fats  into  fatty  acids  and  glycerin.  The  fatty  acids 
remain  in  the  necrotic  areas,  and  the  glycerin  is  absorbed 
into  the  circulation  and  excreted  by  the  urine.  The  original 
test  was  consequently  devised  to  demonstrate  glycerin  in  the 
urine  by  the  production  of  glycerosazone  crystals. 

The  theory  was  subsequently  considerably  altered — the 
tests  A  and  B  were  abandoned  and  replaced  by  a  single 
process  known  as  test  C.  The  nature  of  the  crystals  found 
in  the  test  was  reconsidered,  and  the  clinical  significance 
given  to  the  results  of  the  test  was  modified.  The  present 
theory  of  the  reaction  appears  to  be  as  follows.  The  crystals 
produced  result  from  the  presence  of  a  sugar  complex, 
which  on  hydrolysis  with  hydrochloric  acid  yields  a  substance 
having  the  reaction  of  a  pentose.  The  crystals,  therefore, 
of  a  positive  reaction  are  supposed  to  be  pentosazone 
crystals  (see  also  page  303).  The  pancreas  is  stated  to 
contain  five  times  as  much  pentose  as  any  other  organ  in 
the  body,  and  when  partial  disintegration  of  the  pancreas 
occurs  as  the  result  of  disease  the  Cammidge  crystals  will  be 
obtained  in  the  test.  On  this  theory  a  mere  blocking  of  the 
pancreatic  secretion  will  not  yield  a  positive  urinary  reaction, 
nor  will  fibrosis  of  the  pancreas  apart  from  active  disease. 
Cammidge  is  consequently  of  opinion  that  a  positive  reaction 
is  evidence  of  active  degeneration,  such  as  occurs  in  acute 
liEemorrhagic  or  in  chronic  pancreatitis,  and  a  negative  re- 
action contra-indicates  active  degeneration,  but  does  not 
exclude  old  pancreatitis  nor  malignant  disease  of  the  pancreas. 
In  75  per  cent,  of  malignant  cases  the  reaction  is  negative. 

Cammidge  does  not  claim  that  the  test  is  always  positive  in 
cases  of  pancreatitis,  nor  that  a  negative  test  definitely 
excludes  pancreatitis,  but  insists  that  the  result  must  be  con- 
sidered in  conjunction  with  the  clinical  findings,  with  other 
urinary  tests,  and  in  particular  with  an  examination  of  the 
fasces  (see  page  321). 

A  considerable  experience  of  the  test  has  not  convinced 
me  of  its  practical  value  in  diagnosis,  and  there  can  be  no 
doubt  that  positive  reactions  of  marked  intensity  may  be 
obtained  in  a  considerable  variety  of  affections.  At  the  same 
time  it  must    be  allowed  that   an  abundant   deposit   of   the 


SPECIAL  INVESTIGATIONS  OF  THE  UBINE,  ETC.  275 

crystals  is  an  interesting  abnormality  and  lias  some  meaning, 
however  cryptic  this  may  be  at  the  present. 

Test  C  is  performed  as  follows : — 

Preliminary. — Filter  a  portion  of  a  24  hours  specimen  of 
urine. 

Test  for  albumin. — If  albumin  is  present  in  amount  more 
than  a  mere  trace,  measure  out  50  c.c.  of  the  nitrate,  and  add 
a  few  drops  of  acetic  acid.  Boil.  Cool.  Filter,  and  make  up 
nitrate  to  50  c.c. 

Test  for  sugar. — Either  Fehling's  or  Nylander's  test,  care- 
fully performed,  must  be  completely  negative.  If  there  is  any 
reduction  on  standing  about  50  c.c.  of  the  albumin-free  urine 
must  be  mixed  with  yeast,  fermented  for  from  12  to  24  hours, 
and  filtered. 

Stage  1. — Measure  20  c.c.  of  the  clear  albumin-  and  sugar- 
free  nitrate  into  a  small  flask  with  an  inverted  filter  funnel 
placed  in  its  mouth  as  a  condenser. 

Add  1  c.c.  of  strong  HC1. 

Boil  on  the  sand  bath  for  10  minutes  from  the  commence- 
ment of  ebullition.  The  boiling  should  not  be  too  vigorous,  and 
the  flame  should  be  turned  low  for  the  greater  part  of  the  time. 

Stage  2. — Cool  under  the  tap. 

Make  up  contents  to  20  c.c.  with  distilled  water. 

Slowly  add  4  grammes  of  lead  carbonate  ;  shake  gently  at 
first  and  more  thoroughly  later.  Stand,  and  shake  occasion- 
ally until  no  more  gas  comes  off. 

Filter  through  a  paper  moistened  with  distilled  water. 

Stage  3. — Add  4  grammes  of  powdered  tribasic  lead  acetate. 

Shake  thoroughly  for  some  minutes  and  allow  to  stand. 

Filter  through  a  moistened  filter  paper. 

Stage  4. — To  the  clear  and  almost  colourless  filtrate  add 
2  grammes  of  powdered  sodium  sulphate. 

Shake  thoroughly  for  several  minutes. 

Bring  slowly  up  to  the  boiling  point  on  a  sand  bath,  shaking 
from  time  to  time. 

The  excess  of  lead  is  removed  at  this  stage,  and  it  is 
important  that  the  shaking  and  heating  should  be  done  care- 
fully. 

Stage  5. — Cool  under  the  tap  and  filter. 

Measure  10  c.c.  of  clear  filtrate. 

Make  them  up  to  18  c.c.  with  distilled  water. 

18—2 


276  CLINICAL   PATHOLOGY. 

Add  0*8  gramme  of  phenyl  hydrazine  hydrochlorate, 
2  grammes  of  powdered  sodium  acetate,  and  1  c.c.  of  50  per 
cent,  acetic  acid. 

Boil  in  a  flask  with  a  funnel  condenser  on  the  sand  bath  for 
10  minutes  from  the  commencement  of  ebullition.  Do  not 
boil  too  vigorously. 

Filter  hot  through  a  filter  paper  moistened  with  boiling 
distilled  water  into  a  15  c.c.  measure.  Should  the  filtrate  fail 
to  reach  the  15  c.c.  mark,  make  up  to  15  c.c.  with  hot  distilled 
water.  Stand  for  from  4  to  5  hours  or  longer  at  room 
temperature  or  in  the  ice  chest. 

Examine  the  filtrate  for  the  appearance,  solubility,  and 
amount  of  crystal  formation. 

The  typical  crystals,  examined  under  the  microscope,  are  of 
the  osazone  type  and  more  circular  and  tuft-like  than  glucosa- 
zone  crystals  (see  Plate  XI.).  Eun  under  the  cover-slip  33  per 
cent,  sulphuric  acid  ;  the  crystals  should  be  dissolved  in  from 
10  to  15  seconds.  The  crystals  have  to  be  distinguished  from 
the  coarse  yellow  needles  which  may  be  deposited  if  the 
excess  of  lead  was  not  removed  in  stage  4.  In  a  strongly 
positive  reaction  the  deposit  of  crystals  may  occupy  half  the 
bulk  of  the  filtrate.  In  a  completely  negative  reaction  the 
filtrate  remains  clear. 

In  all  cases  the  deposit  should  be  examined  microscopically 
and  chemically. 

The  Bacteriology  of  the  Urino -genital  Tract. 

The  variety  of  pathogenic  organisms  commonly  met  with  in 
the  urino -genital  tract  is  not  very  great,  and  in  general  the 
bacteriological  examinations  should  be  conducted  on  the  lines 
indicated  in  the  section  dealing  with  bacteriology,  where 
a  further  account  of  the  organisms  will  be  found.  The  special 
precautions  to  be  attached  to  the  investigation  of  this  tract 
are  considered  under  the  heading  of  each  organism. 

The  gonococcus. — In  the  male  the  recognition  of  the 
gonococcus  in  a  purulent  urethral  discharge  is  commonly 
a  simple  matter,  and  in  the  very  great  majority  of  cases  the 
causative  organism  of  an  acute  urethritis  is  the  gonococcus. 
Occasionally  some  other  organism  is  the  cause  of  the  inflam- 
mation, and  in  some  cases  a  discharge  of  pus  from  the  urethra 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  277 

has  some  external  origin,  as  for  example  a  prostatic  abscess, 
and  may  be  caused  by  the  colon  bacillus  or  a  staphylococcus. 
In  cases  of  chronic  gleet,  so  long  as  there  is  a  considerable 
urethral     discharge,     gonococci     commonly     remain     fairly 
numerous.       Gonococci  may  in  exceptional  cases  produce  a 
urethritis  associated  with  the  causative  organism  which  lasts 
for   years.     Not  infrequently  a   history   will   be   given    of   a 
urethritis  which  cleared  up  some  years  previously  and  which 
has  again  recurred.     If  a  thick  purulent  discharge  loaded  with 
gonococci  be  found,  it  is  extremely  probable  that  the  patient 
has  exposed  himself  to  a  second  infection.     The  latency  of  the 
gonococcus  in  the  urethra  is  a  most  important  question,  and 
the  examination  of  the  urethra  for  evidence  of  such  infection 
is  a  procedure  requiring  the  co-operation  of  the  surgeon  and 
the  pathologist.     If  a  slight  morning  discharge  is  present,  or 
if  pus  cells  and  prostatic  threads  are  found  in  the  morning 
specimen   of    urine,    or   even   if   the   patient   can   detect   no 
discharge  at  all,  the  urethra  may  still  be  infective.      In  all 
cases  in  which  the  patient  seeks  advice  as  to  infectivity,  and 
particularly  with  a  view  to  matrimony,  a  thorough  search  has 
to  be  made  for  the  gonococcus  as  well  as  for  clinical  evidence 
of  urethral   inflammation.      The    patient    should    come  for 
examination  in  the  early  morning  under  instructions  to  hold 
his  water  until  he  has  been  investigated.     The  orifice  of  the 
urethra   should   be  first   examined,  and  if   any  discharge  is 
found  film  preparations  should  be  made  from  it  and  cultures 
taken  on  serum  agar.      The  urine  should  then  be  passed  and 
examined  by  the  naked  eye  for  prostatic  threads.     The  urine 
should  then  be  centrifuged  and  the  deposit  searched  for  pus 
and  epithelial  cells,  and  if  these  are  present  gonococci  should 
be  carefully  looked  for.      The  anterior  urethra  should  next  be 
thoroughly  irrigated  with  sterile  water.     The  patient  should 
then  be  placed  on  his  hands  and  knees  and  vigorous  massage 
of  the  prostate  performed.     Several  minims  of  clear  or  turbid 
prostatic  fluid  can  often  be  made  to  flow  out  from  the  urethra, 
and  this  is  collected  and  examined  for  the  presence  and  nature 
of  the  cells  and  organisms.     The  urinary  examination  alone 
for  gonococci  should  never  be  depended  upon,  since  it  is  only 
exceptionally  that  gonococci  can  be  recognised  in  a  urinary 
deposit.     If  a  purulent  morning  discharge  is  absent,  if  there 
is  no  pus  in  the  urine,  and  if  the  prostatic  fluid  is  clear  and 


278  CLINICAL   PATHOLOGY. 

contains  only  mononuclear  cells,  the  patient,  in  the  absence  of 
clinical  signs  or  symptoms,  is  probably  free  from  gonorrhoea. 
If  any  of  these  abnormalities  are  present  the  gonococcus  may 
or  may  not  be  found.  All  cases  of  residual  urethritis  or 
prostatitis  after  gonorrhoea  are  by  no  means  due  to  the 
gonococcus.  Failure  to  find  the  gonococcus  at  one  examina- 
tion does  not,  however,  exclude  the  possibility  of  infectivity. 
A  further  search  should  be  made  and  conducted  on  the  same 
lines,  but  after  the  passage  of  a  large-sized  sound  and  the 
irrigation  of  the  urethra  with  strong  silver  solution.  The 
inflammatory  cells  resulting  from  these  procedures  should 
be  searched  for  gonococci.  If  no  gonococci  are  found  the 
patient  is  as  certainly  free  from  infection  as  a  reasonably 
careful  investigation  can  prove. 

The  examination  of  the  female  genito-urinary  tract  for 
gonococci  is  less  commonly  successful,  even  in  the  acute  stage, 
and  the  special  precautions  to  be  adopted  have  been  previously 
described  (page  122). 

The  spirochaeta  pallida. — The  demonstration  of  the 
spiroclaeta  pallida  has  been  described  under  the  account  of . 
that  organism,  and  it  is  only  reasonable  that  the  clinical 
diagnosis  of  every  genital  chancre  should  be  controlled  by  a 
bacteriological  examination.  In  dealing  with  a  chancre  of  the 
penis  help  in  obtaining  the  clear  serous  exudate  from  the  base  of 
the  cleaned  ulcer  may  often  be  derived  from  the  use  of  a  small 
Bier's  cup.  The  cup  should  have  an  orifice  of  about  the  size 
of  a  sixpence,  the  edge  should  be  lightly  smeared  with 
vaseline,  the  rubber  ball  flattened  and  the  cup  firmly  placed 
over  the  ulcer.  On  releasing  the  ball  a  quantity  of  serum 
is  often  sucked  out,  and  the  spirochgetes  present  in  this 
may  be  very  numerous.  Spirochgetes,  other  than  the 
syphilitic  organisms,  are  not  commonly  present  in  genital 
chancres,  but  those  of  the  refringens  type  may  be  very 
numerous  in  extensively  ulcerated  sores  of  the  "  soft " 
variety. 

Ducrey's  bacillus. — To  this  organism  is  accredited  the 
causation  of  the  soft  chancre.  The  bacillus  may  be  found  in 
the  ulcer  and  in  the  buboes.  It  has  been  inoculated  experi- 
mentally from  the  chancre  in  human  beings,  but  the  disease 
has  not  been  reproduced  in  animals.  The  organism  is  a  very 
small  Gram-negative  bacillus,  which   does  not  grow  on  the 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  279 

ordinary  media.  On  blood  agar  it  grows  in  small,  round, 
glistening  colonies. 

The  tubercle  bacillus. — The  method  for  the  detection  of 
tubercle  bacilli  in  the  urine  has  been  described  in  Chapter  XL, 
page  157.  A  24  hours  specimen  of  urine  is  preferable,  but  if 
the  bacilli  are  abundant  they  may  readily  be  found  in  a 
catheter  specimen.  It  may  be  necessary  to  determine  if  one 
or  both  kidneys  are  tuberculous,  and  the  question  is  sometimes 
solved  by  an  examination  of  ureteric  specimens  from  the  two 
sides.  Tubercle  bacilli  may  be  found  on  one  side  only  or  on 
both  sides,  but  a  negative  examination  is  not  very  weighty 
evidence  against  tuberculous  nephritis.  Such  specimens 
should  also  be  examined  for  the  presence  of  pus,  care  being 
taken  not  to  confuse  the  small  round  mononuclear  epithelial 
cells  with  pus  cells.  If  no  pus  is  present  the  kidney  is 
probably  healthy.  Whatever  the  nature  of  the  urinary 
specimen  examined,  a  single  failure  to  find  the  bacilli  does 
not  greatly  outweigh  clinical  evidence.  Two  or  more 
examinations  may  be  required  before  the  bacilli  can  be 
found  in  cases  where  they  are  scanty.  It  is  not  an  infrequent 
experience  to  spend  half  an  hour  over  a  slide  and  to  find 
only  one  group  of  bacilli.  It  is  practically  useless  to  examine 
any  specimen  for  tubercle  bacilli  if  no  pus  is  present ; 
even  if  acid-fast  bacilli  are  found,  in  the  absence  of  pus 
there  will  be  grave  doubt  as  to  their  nature. 

The  colon  bacillus. — A  catheter  specimen,  carefully 
taken  after  cleaning  the  urethral  orifice,  is  essential  for  the 
detection  of  colon  bacilli  in  the  female.  In  the  male  a 
catheter  specimen  is  preferable  but  not  essential.  If  there 
is  an  objection  to  the  passage  of  a  catheter  the  glans 
penis  should  be  thoroughly  cleansed,  and  the  patient  should 
be  instructed  to  pass  the  first  portion  of  urine  into  an 
ordinary  receiver,  and  the  next  portion  into  a  sterile  wide- 
mouthed  flask  fitted  with  a  wool  plug. 

When  the  specimen  is  obtained  proceed  as  follows : — 

Pour  approximately  3  to  4  c.c.  of  urine  into  a  broth  tube. 
Set  aside  the  remainder  of  the  specimen  for  further 
investigation. 

Incubate  the  culture  at  37°  C.  for  from  12  to  24  hours. 

Examine  the  broth  culture  for  general  turbidity,  and 
microscopically  for  the  presence  of  the  bacilli, 


280  CLINICAL   PATHOLOGY. 

If  a  growth  of  bacilli  has  taken  place,  prepare  two  Petri  dish 
plate  cultures,  one  of  which  contains  agar  and  the  other 
MacConkey's  medium. 

Take  a  platinum  loop  of  the  broth  culture  and  make  a 
series  of  streaks,  first  on  the  agar  plate  and  next  (without 
recharging  the  loop)  on  the  MacConkey  plate. 

Incubate  the  plate  cultures  and  the  original  broth  culture 
till  the  next  morning. 

Examine  both  plates,  and  in  particular  the  MacConkey 
plate.  If  large  red  colonies  are  present  on  the  MacConkey 
plate  and  all  are  apparently  of  the  same  nature,  subculture 
from  them  into  the  following  media  : — Litmus  milk.  Neutral 
red  broth.  Broth.  Gelatin  slope.  Litmus  dextrose.  Litmus 
mannite.  Litmus  lactose.  Incubate  the  sub-cultures  until 
the  next  morning,  but  remember  to  place  the  gelatin  slope 
in  a  separate  crate  at  a  temperature  of  from  18°  to  22°  C. 

Examine  the  sub-cultures  for  the  following  changes : — 

Acid  and  clot  in  milk ;  green  fluorescence  in  neutral  red 
broth ;  indole  in  broth,  as  shown  by  the  production  of  a  rose- 
pink  colour  on  the  addition  of  nitric  acid  ;  growth  on  gelatin 
without  liquefaction  ;  acidity  and  gas  formation  in  the  three 
litmus  carbohydrate  media. 

If  all  these  changes  are  present  the  organism  is  the  colon 
bacillus. 

If  the  changes  have  not  yet  occurred,  reincubate  the  tubes 
for  another  24  or  48  hours,  or  longer  if  necessary. 

The  agar  plate  should  also  be  examined  for  the  presence  of 
cocci  in  addition  to  the  bacilli.  Colonies  of  staphylococcal  or 
streptococcal  nature  should  be  examined  microsopically,  and 
if  cocci  are  present  sub-cultures  should  be  made  in  broth  or 
on  agar  and  the  cultural  character  of  the  organisms  further 
investigated. 

No  cocci  will  be  found  on  the  MacConkey  plate. 

If  bacilli  of  the  colon  type  are  found  on  the  agar  plate  and 
no  growth  has  taken  place  on  the  MacConkey  plate,  this 
should  be  reinoculated  from  the  original  broth  culture.  If 
no  growth  occurs  after  reinoculation,  colon  bacilli  are 
absent. 

If  colonies  of  more  than  one  type  or  colour  are  present  on 
the  MacConkey  plate,  a  characteristic  discrete  colony  of  each 
type  must  be  transferred  to  broth  and  sub-cultures  from  each 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  281 

broth  tube  made  on  the  following  day  into  the  series  of  media 
given  for  the  colon  bacillus. 

The  catheter  specimen  of  urine  must  on  every  occasion  be 
examined  generally  as  well  as  bacteriologically .  Very  little  infor- 
mation is  derived  from  the  cultivation  of  a  colon  bacillus  from  the 
urine,  and  such  information  as  is  given  may  be  totally  misleading. 
Colon  bacilli  in  the  urine  are  not  by  any  means  the  necessary 
explanation  of  an  obscure  fever  or  even  of  definite  urinary 
symptoms.  All  bacterial  investigations  must  be  considered  in 
conjunction  with  the  general  examination  of  the  urine  and  the 
clinical  condition  of  the  patient.  The  further  examinations  of 
the  urine  to  be  invariably  made  are  simple,  and  concerned 
with  the  appearance,  the  reaction,  the  presence  and  amount  of 
albumin,  and  the  nature  of  the  deposit. 

The  following  types  of  urinary  infection  may  be  recog- 
nised : — 

In  the  latent  type  colon  bacilli  may  exist  in  the  urine  of 
men  or  women,  but  more  frequently  the  latter,  without  pro- 
ducing any  signs  or  symptoms  of  disease  or  any  changes  in 
the  urine.  In  such  cases  the  urine  is  normal,  no  bacilli  are 
seen  in  the  centrifuged  deposit,  and  a  growth  of  the  colon 
bacillus  is  obtained  on  culture.  This  class  of  case  constitutes 
the  "colon  carrier."  The  condition  is  liable  to  pass  into  the 
acute  inflammatory  stages,  particularly  if  pregnancy  or  some 
other  complication  arises. 

In  the  acute  type  the  patient  has  marked  vesical  and  often 
renal  symptoms,  has  high  fever,  sometimes  with  rigors,  and 
may  present  all  the  aspects  of  extremely  serious  illness.  The 
urine  is  turbid  on  passing,  the  turbidity  being  due  to  the 
enormous  numbers  of  bacilli.  Pus  cells  are  present,  but  they 
may  be  scanty,  and  red  cells  may  be  found.  A  trace  of  albumin 
is  present.  Such  patients  practically  always  recover  from  this 
severe  stage  under  treatment  and  often,  apparently,  in  spite  of 
treatment.  They,  however,  frequently  pass  into  the  chronic 
type. 

In  the  chronic  type,  which  may  persist  for  months  and 
may  be  apparently  chronic  from  the  onset,  some  other  pre- 
disposing cause,  such  as  pregnancy,  obstinate  constipation, 
hemorrhoids,  or  an  ischio-rectal  abscess,  is  often  present.  The 
urinary  symptoms  are  subacute  or  absent,  a  mild  and  inter- 
mittent pyrexia  is  present,  and  the  urine  is  acid,  containing  a 


282  CLINICAL   PATHOLOGY. 

trace  of  albumin  and  a  considerable  naked-eye  deposit,  due 
to  pus  and  bacilli. 

The  presence  of  colon  bacilli,  therefore,  in  a  culture  taken 
from  the  urine  is  insufficient  evidence  that  the  bacilli  are 
actively  producing  disease,  and  if  no  pus  is  present  it  is  almost 
certain  that  they  are  not.  Even  if  pus  and  bacilli  are  present 
it  does  not  follow  that  the  ultimate  diagnosis  has  been  reached. 
Infection  by  the  colon  bacillus  is  readily  superimposed  upon 
some  other  lesion,  such  as  that  of  tuberculosis  or  calculus. 
The  bacillus  proteus  is  in  such  instances  often  present  in 
addition. 

In  all  cases  of  coli  infection  of  the  urine  associated 
with  a  considerable  degree  of  pyuria  it  is  a  wise  pre- 
caution to  search  the  urine  for  tubercle  bacilli  also. 

The  treatment  of  coli  infections  by  autogenous  vaccines  is 
worthy  of  trial  in  selected  cases. 

The  bacillus  proteus. — The  examination  for  this  organism 
is  conducted  in  exactly  the  same  manner  as  that  for  the  colon 
bacillus.  The  bacilli  grow  well  on  the  MacConkey  plate,  and 
are  recognised  by  the  yellow  colour  of  their  colonies,  the  more 
slimy  nature  of  their  growth,  and  their  somewhat  character- 
istic and  offensive  odour.  The  cultural  character  which  mainly 
distinguishes  them  from  the  colon  bacillus  is  the  power  of 
liquefying  gelatin.  Bacilli  which  in  other  respects  are  cul- 
turally identical  with  the  colon  bacillus  may  grow  in  yellow 
colonies  on  the  MacConkey  plate.  Pure  infections  of  the 
urinary  tract  by  B.  proteus  occur,  but  the  organism  is  more 
frequently  found  in  association  with  other  bacteria  and  other 
lesions  of  the  tract,  such  as  neoplasm  of  the  bladder  or  renal 
calculus.  In  cases  of  proteus  infection  the  urine  is  commonly 
alkaline. 

The  typhoid  bacillus. — This  organism  is  examined  for  by 
the  same  routine.  The  cultural  characters  are  given  under 
the  description  of  the  bacillus.  The  bacillus  should  be  further 
identified  by  serum  agglutination  tests,  using  both  the  serum 
of  the  patient  and  that  from  a  known  case  of  typhoid  fever. 

The  typhoid  bacillus  may  exist  in  the  urine  without  pro- 
ducing symptoms,  and  in  such  cases  is  a  dangerous  source  of 
infection  to  others. 

Bacilluria  following  typhoid  fever  is  common,  but  is  in  the 
majority  of  cases  due  to  the  B.  coli. 


SPECIAL  INVESTIGATIONS  OF  THE  URINE,  ETC.  283 

Staphylococci :  Streptococci :  Diplococci.— Any  of  the 

ordinary  pyogenic  cocci  may  be  found  in  the  urine,  but  in  the 
great  majority  of  cases  they  come  from  the  lower  part  of  the 
tract  and  are  of  little  importance. 

The  specimen  for  examination  mast  be  a  catheter  specimen, 
and  preferably  should  be  withdrawn  after  thorough  irrigation 
of  the  urethra  with  sterile  water. 

The  urine  should  be  first  added  to  broth,  and,  if  the  growth  in 
the  broth  tube  appears  to  be  purely  coccal,  plate  cultures 
should  be  made  on  two  agar  Petri  dishes.  The  colonies  on 
agar  are  examined  in  the  usual  way.  Any  of  the  three 
varieties  of  staphylococci  may  be  present,  and  may  produce 
a  cystitis  associated  with  a  purulent  and  often  alkaline  urine. 
Staphylococcal  cystitis  is  rarely  primary,  and  is  more  often 
superimposed  upon  some  other  lesion,  such  as  a  urethral 
stricture  or  prostatic  enlargement. 

Streptococci  are  less  frequently  met  with,  if  the  urethral 
lavage  has  been  thorough.  A  long  chained  streptococcus,  of 
low  pathogenicity  to  animals,  is  the  variety  most  commonly 
found. 

Pneumococci  in  the  urine  are  often  recorded  and  extremely 
rarely  found.  The  organism  commonly  confounded  with 
the  pneumococcus  is  a  paired  coccus  met  with  frequently 
in  the  urinary  tract.  This  diplococcus  is  Gram-positive  and 
morphologically  resembles  the  pneumococcus,  but  is  readily 
distinguished  by  its  cultural  characters.  The  coccus  grows 
abundantly  in  all  media,  producing  an  acid  reaction.  Litmus 
milk  is  characteristically  clotted  and  completely  decolorised 
in  about  12  hours.  Abundant  growth  in  pin-point  colonies 
occurs  on  gelatin  without  liquefaction.  The  coccus  is  non- 
pathogenic to  mice.  The  organism  is  more  commonly  met 
with  in  specimens  from  women  and  children  than  from  men, 
and  in  the  majority  of  cases  gives  rise  to  no  symptoms.  It 
may,  however,  be  found  in  pure  culture  in  cases  of  cystitis  or 
pyelitis,  and  it  has  been  recovered  from  the  general  circulation. 

Other  organisms. — M.  melitensis  can,  in  the  great  majority 
of  cases  of  Malta  fever,  be  isolated  from  the  urine  during  the 
early  period  of  the  disease.  The  cocci  grow  slowly  and  in 
small  colonies  upon  agar. 

Bacilli  of  the  influenza  type  are  occasionally  met  with  in 
association  with  a  purulent  urethral  discharge  in  which  no 


284  CLINICAL   PATHOLOGY. 

gonococci  can  be  found.  Such  unusual  infections  may  follow 
sexual  intercourse. 

Diphtheroid  bacilli  are  frequently  found  in  the  urine,  and 
are  practically  always  derived  from  the  lower  urethra.  The 
appearance  of  these  organisms  in  the  cultures  is  evidence  of 
urethral  contamination.     The  bacilli  are  apparently  harmless. 

In  conclusion,  any  of  the  organisms  of  the  colon-typhoid 
group  not  previously  mentioned  may  be  found  in  the  urine,  and 
bacilli  of  this  group  which  are  difficult  to  exactly  classify  are 
fairly  frequently  met  with. 


SECTION  Y. 

THE   ALIMENTARY   SYSTEM. 

CHAPTER  XX. 

The  Mouth— The  Stomach. 

CHAPTER  XXI. 

The  Pancreas — The  Liver — The  Spleen — The  Peritoneum. 

CHAPTER  XXII. 
The  Faeces. 

CHAPTER  XXIII. 

The  Parasitology  of  the  Faeces. 


CHAPTER  XX. 

the  mouth the  stomach. 

The  Mouth. 

The  laboratory  investigations  of  the  buccal  cavity  are 
mainly  bacteriological  in  nature.  Examinations  of  the 
chemical  composition  of  the  saliva  or  of  its  cellular  content 
have  very  little  practical  bearing. 

Oral  sepsis  and  intestinal  toxaemia  have  borne  the  odium 
of  all  the  ills  which  man  may  suffer.  There  can  be  no 
question  that  a  thoroughly  septic  condition  of  the  mouth, 
leading  to  an  absorption  of  offensive  pus,  may  set  up  con- 
siderable disturbance,  of  which  the  most  direct  effect  is  gastric 
or  intestinal  in  nature.  More  remote  lesions  due  to  the 
absorption  of  toxins  into  the  circulation  are  also  possible,  but 
difficult  to  prove.  Of  these  the  most  probable  is  a  variety  of 
osteo-arthritis,  which  more  particularly,  attacks  the  phalanges, 
and  is  very  commonly  associated  with  some  local  septic  lesion 
such  as  pyorrhoea  alveolaris.  The  bacteriological  investi- 
gation of  the  mouth  is  reasonable  in  such  cases,  and  is 
occasionally  profitable.  The  practitioner  should,  however,  be 
warned  against  the  indiscriminate  use  of  vaccines  prepared 
from  organisms  of  the  buccal  cavity.  Nothing  is  more  simple 
than  the  preparation  of  such  a  vaccine  for  any  and  every 
condition,  and  nothing  would  be  more  futile  were  it  not  for 
the  mental  effect  of  a  hypodermic  injection  upon  a  confiding 
patient.  Pernicious  anaemia,  the  leukaemias,  arthritis  of  every 
variety  including  gout,  and  almost  every  form  of  gastric  and 
intestinal  disorder,  have  been  attributed  to  oral  sepsis.  I  have 
even  known  prolapse  of  the  kidney  explained  by  the  same 
cause.  We  can  be  certain  at  any  rate  of  this,  that  all  such 
diseases  readily  develop  in  the  absence  of  any  oral  sepsis,  and 
if  present  may  progress  in  spite  of  its  cure.  These  views  are 
not  of  course  opposed  to  the  rational  treatment  of  a  septic 
mouth,  nor  can  it  be  imagined  that  the  absorption  of  pus  from 
infected  gums  is  anything  but  harmful. 


THE   MOUTH— THE    STOMACH.  287 

Pyorrhoea  alveolaris  is  readily  recognised  by  the  exudation 
of  pus  from  the  sockets  of  the  teeth.  In  severe  cases  the 
majority  of  the  teeth  are  loose  and  almost  floating  in  a 
purulent  bed.  The  teeth  themselves  may  show  little  or  no 
signs  of  caries.  In  order  to  make  a  cultural  examination  the 
mouth  should  be  well  washed  out  with  clean  and  preferably 
sterile  water,  and  a  loop  of  pus  as  it  exudes  after  pressure  on 
the  crown  of  a  tooth  be  taken  in  a  platinum  wire.  If  little 
discharge  is  present,  a  sterile  wool  swab  similar  to  that  used 
for  diphtheria  cases  may  be  rubbed  against  the  root  of  the 
tooth.  If  it  is  advisable  to  extract  a  tooth,  cultures  may  be 
taken  from  the  socket.  The  platinum  wire  or  swab  should 
be  rubbed  over  the  surface  of  an  agar  or  nasgar  slope,  or 
preferably  by  a  series  of  streaks  over  two  agar  plates.  Films 
of  the  pus  should  also  be  made.  The  cultures  are  incubated 
till  the  next  morning,  and  the  nature  of  the  colonies  investi- 
gated by  the  hand  glass  and  microscopically.  Sub-cultures 
should  be  made  from  each  variety  of  colony  on  the  plate  and 
their  full  cultural  characters  investigated.  The  predominant 
organism  in  the  pus  films  and  in  the  plate  cultures  should  be 
noted.  In  the  films  of  pus  numerous  spirilla  and  fusiform  or 
beaded  bacilli  can  often  be  seen,  but  they  do  not  grow  in  the 
cultures.  If  a  vaccine  is  required  it  should  be  made  from  the 
predominant  organism  of  greatest  virulence,  and  in  a  case  of 
doubt  a  mixed  vaccine  can  be  prepared.  The  organism  most 
commonly  found  and  most  frequently  predominant  in  the 
plate  cultures  is  a  short-chained  streptococcus  of  the  brevis 
type.  This  organism  grows  in  short  chains  of  6  to  10 
members,  and  very  constantly  acidifies  and  clots  litmus  milk. 
It  appears  to  be  more  closely  allied  to  the  pneumococcus  than 
to  the  streptococcus  pyogenes,  but  differs  from  both  in  being 
practically  non-virulent  to  mice  and  rabbits.  Other  organisms 
which  may  be  obtained  are  other  varieties  of  streptococci, 
micrococcus  catarrhalis,  and  less  commonly  the  pneumococcus. 
Staphylococci  are  usually  present  in  addition,  and  almost  any 
of  the  pathogenic  organisms,  including  the  bacillus  coli,  may 
be  occasionally  isolated. 

Thrush  is  an  infection  of  the  buccal  cavity  by  the  oidium 
albicans.  It  is  common  in  children,  but  occurs  also  in  adults, 
and  particularly  in  febrile  patients  the  care  of  whose  mouths 
has  been  neglected.     A  white,  flaky  membrane  is  present  and 


288  CLINICAL   PATHOLOGY. 

may  spread  over  the  entire  buccal  cavity,  including  the  palate 
and  the  tongue.  The  membrane  is  usually  detachable  with- 
out leaving  a  raw  surface.  In  children  the  membrane  has  to 
be  distinguished  from  particles  of  milk  left  in  the  mouth  after 
feeding.  In  films  made  from  the  membrane  an  abundant 
interlacing  mycelium  is  usually  found,  together  with  a  few  of 
the  yeast-like  oval  cells.  In  cultures  the  cellular  element  of 
the  organism  may  predominate. 

The  condition  is  readily  cured  by  ordinary  antiseptic  treat- 
ment. 

Diphtheria  produces  a  membrane  which  is  most  commonly 
confined  to  one  tonsil,  and  is  removed  with  difficulty,  leaving 
a  bleeding  surface.  There  may  be  little  constitutional  dis- 
turbance. The  methods  of  taking  and  examining  swabs  and 
cultures  from  suspected  cases  of  faucial  diphtheria  have 
been  already  described  (p.  129).  The  organisms  are  often 
difficult  to  identify  in  swab  preparations,  but  are  readily 
recognised  after  from  8  to  12  hours'  incubation  on  blood 
serum. 

Vincent's  angina  consists  in  an  ulcerative  stomatitis, 
often  with  membrane  formation.  The  condition  is  most 
frequently  met  with  in  young,  ill-nourished  children,  and  is 
accompanied  by  severe  general  symptoms.  In  films  made 
from  the  ulcerated  surface  the  organisms  associated  with 
Vincent's  name  are  very  numerous.  Pus  cells  may  be  scant}' 
and  lying  on  a.  background  of  innumerable  spirilla  of  varying 
shape  and  size.  The  fusiform  bacilli  are  less  numerous  and 
of  similarly  variable  size,  some  being  stout  and  cigar-shaped, 
others  long,  thin,  and  beaded.  It  is  by  no  means  certain  that 
these  organisms  are  the  essential  cause  of  the  stomatitis. 
They  are  very  numerous  in  almost  any  septic  mouth,  and  in 
Vincent's  angina  other  pyogenic  organisms,  such  as  strepto- 
cocci and  staphylococci,  are  present  in  addition. 

Follicular  tonsilitis  is  characterised  by  swelling  of  one 
or  both  tonsils,  and  by  numerous  small,  round  yellow  spots 
of  suppuration  in  them.  The  organisms  found  in  the  pus 
are  commonly  staphylococci  or  streptococci.  Streptococcal 
inflammation  may  produce  swelling  and  hyperemia  of  the 
tonsil  only,  and  these  organisms  are  usually  associated  with 
the  angina  of  scarlet  fever  as  well  as  with  the  tonsilitis  which 
almost   invariably  precedes   by  10   days   or   a   fortnight   an 


THE    MOUTH— THE    STOMACH.  289 

attack  of  rheumatic  fever.  The  streptococcus  associated  with 
scarlet  fever  is  often  a  long-chained  one,  while  that  obtained 
from  the  rheumatic  tonsil  is  usually  of  the  brevis  type. 
Streptococci  may  also  set  up  a  membranous  tonsilitis  closely 
resembling  that  of  diphtheria,  but  usually  associated  with 
severer  constitutional  disturbance.  The  distinction  between 
the  two  conditions  is  readily  made  on  bacteriological  grounds. 

The  Stomach. 

Laboratory  investigations  into  the  functions  of  the  stomach 
are  mainly  chemical  in  nature  and  are  principally  concerned 
with  an  analysis  of  the  gastric  juice.  The  diagnosis  of  a 
condition  associated  with  gastric  symptoms  as  "  dyspepsia " 
has  little  real  meaning  and  carries  us  no  further  than  a 
diagnosis  of  "  anaemia  "  in  other  conditions.  A  careful 
analysis  of  the  gastric  contents  associated  with  the  clinical 
examination  of  the  patient  enables  us  in  the  great  majority  of 
cases  to  recognise  the  condition  present  and  the  treatment 
which  should  be  adopted.  Of  all  the  methods  of  investigation 
into  the  composition  of  the  gastric  juice,  that  of  the  "  test 
meal"  analysis  is  the  most  important. 

The  Test  Meal. — The  sole  difficulty  in  the  procedure  lies 
in  the  withdrawal  of  the  test  meal,  in  reality  a  very  simple 
manoeuvre.  The  passage  of  an  oesophageal  tube  is  uncomfort- 
able, but  free  from  pain  or  danger  in  careful  hands.  The 
objection  of  the  patient  to  its  passage  varies  inversely  with 
the  tact  and  confidence  of  the  practitioner. 

The  meaning  of  the  results  obtained  by  the  analysis  of  gastric 
contents  will  be  discussed  later.  If  the  various  precautions 
indicated  below  are  properly  observed,  the  results  of  the  analysis 
may  be  depended  upon.  It  must  be  very  clearly  understood, 
however,  that  these  results  considered  alone  are  of  practically 
no  value  in  diagnosis,  but  must  be  read  in  conjunction  with 
the  signs  and  symptoms  of  the  patient.  It  is  a  most  elementary 
error  to  suppose  that  absence  of  free  hydrochloric  acid  in  a 
test  meal  means  that  carcinoma  of  the  stomach  is  present. 
There  are  even  circumstances  in  which  absence  of  free  acid  is 
opposed  to  the  diagnosis  of  carcinoma.  A  carefully  taken 
history  of  the  case  read  in  conjunction  with  the  analysis  of 
the  test  meal  will  usually  lead  to  a  correct  diagnosis,  but  all 
other  methods  of  investigation  should  be  freely  made  use  of. 

p.  19 


290  CLINICAL   PATHOLOGY. 

The  following  is  the  technique  of  gastric  analysis  : — 

(1)  Lavage  of  the  stomach. — It  is  not  necessary  to  wash 
out  the  stomach  before  giving  the  meal  in  the  majority  of 
cases,  and  indeed  a  more  reliable  result  is  obtained  if  lavage 
is  omitted.  In  cases  of  obvious  dilatation  of  the  stomach  with 
retention  of  food  the  stomach  should  be  emptied  and  washed 
out  with  water  a  few  hours  before  giving  the  meal. 

(2)  The  test  meal  should  be  of  a  simple  character.  It  is 
most  important  that  in  any  series  of  observations  the  same 
type  of  meal  should  be  given,  and  that  in  any  single  observa- 
tion the  nature  of  the  normal  result  obtained  from  such  a 
meal  should  be  known.  The  figures  given  here  are  those 
obtained  from  gastric  contents  after  a  test  meal  which  is 
practically  that  of  Ewald. 

The  meal  consists  of  two  large  cups  of  tea,  with  milk  and 
sugar  if  desired,  and  two  rounds  of  toast  lightly  buttered. 

The  actual  bulk  of  the  meal  is  unimportant,  since  the 
quantity  of  gastric  juice  accommodates  itself  to  the  bulk  of 
material  in  the  stomach.  The  quantity  given,  however,  should 
be  considerable  in  order  to  facilitate  the  subsequent  with- 
drawal. 

(3)  Removal  of  the  gastric  contents. — These  should 
be  removed  exactly  1  hour  after  the  test  meal  has  been  given. 
A  clean  rubber  oesophageal  tube,  the  outside  of  which  has 
been  moistened  in  hot  water,  is  passed  gently  down  the 
oesophagus  into  the  stomach.  The  passage  is  aided  by 
swallowing  movements  on  the  part  of  the  patient,  who  may  be 
lying  in  bed  with  his  head  propped  up  on  pillows,  or  sitting 
up.  Attached  to  the  tube  by  a  glass  junction  should  be  a 
second  piece  of  tubing,  terminating,  if  desired,  in  a  glass 
filter  funnel,  and  the  whole  must  be  sufficiently  long  to  allow 
the  funnel  to  be  held  well  below  the  level  of  the  stomach.  As 
soon  as  the  tube  is  in  the  stomach,  that  is  to  say  has  passed 
freely  at  least  18  inches  beyond  the  teeth,  and  the  resistance 
of  the  stomach  wall  is  felt,  depress  the  funnel  over  a  clean 
specimen  glass  or  other  receptacle  and  tell  the  patient  to 
strain.  The  contents  usually  flow  out  quite  readily.  If 
the  flow  does  not  commence,  alter  slightly  the  position 
of  the  oesophageal  tube  by  withdrawing  it  a  little  or 
pushing  it  further  in.  The  flow  can  sometimes  be  aided  by 
flattening  out  a  few  inches  of  the  tube  with  the  fingers  of  the 


THE   MOUTH— THE    STOMACH.  291 

two  hands  and  releasing  first  the  portion  nearest  the  stomach, 
thus  creating  a  partial  vacuum  to  suck  the  contents  out.  It 
is  never  necessary  to  use  a  pump.  The  straining  movements 
of  the  patient,  aided  if  necessary  by  cautious  pressure  of  the 
hand  on  the  epigastrium,  are  sufficient  to  expel  the  contents.  It 
is  wise  to  have  a  clean  wide  receiver  ready  in  case  the  stomach 
contents  are  vomited  during  the  passage  of  the  tube,  but  this  can 
usually  be  avoided  if  the  patient  is  told  to  refrain  from  straining 
until  the  tube  is  in  its  place.  After  the  contents  have  been  with- 
drawn the  stomach  can  be  washed  out  if  such  treatment  is 
indicated. 

Precautions. — All  medicines  must  be  countermanded  for 
several  hours  previous  to  the  giving  of  the  test  meal.  No 
water  should  be  added  to  the  test  meal,  and  none  passed  down 
the  tube  to  start  the  flow  of  gastric  contents. 

(4)  The  examination  of  the  gastric  contents. — Note 
the  amount  obtained  and  the  appearance.  The  microscopic 
examination  of  slides  made  from  the  contents  is  of  little  value. 
Some  stress  has  been  laid  upon  the  presence  of  bacilli  and 
sarcinse  in  film  preparations,  but  bacteria  seem  to  be  commonly 
present  in  most  gastric  contents.  Sarcinse  are  perhaps  most 
common  in  cases  of  dilated  stomach  with  gastric  fermentation. 

Filter  the  contents  through  a  filter  paper  moistened  with 
distilled  water. 

With  the  filtrate  perform  the  routine  examination  described 
below. 

The  materials  required  are  a  porcelain  evaporating  dish,  a 
burette,  a  beaker,  a  10  c.c.  pipette,  decinormal  caustic  soda 
solution,  Topfer's  solution,  phenol-phthalein  solution,  absolute 
alcohol,  phloroglucin,  and  vanillin. 

Topfer's  solution  consists  of  a  0*5  per  cent,  solution  of 
dimethylamidoazobenzene  in  absolute  alcohol. 

Phenol-phthalein  solution  is  of  0'5  per  cent,  strength  in 
50  per  cent,  alcohol. 

The  procedure  is  as  follows  : — 

(a)  Perform  Giinzburg's  test  for  free  HC1. 

In  a  porcelain  evaporating  dish  place  a  pinch  of  phloro- 
glucin and  a  pinch  of  vanillin. 

Dissolve  in  about  3  c.c.  of  absolute  alcohol. 

Add  about  3  c.c.  of  the  filtered  test  meal. 

Evaporate  slowly  on  a  sand  bath  without  boiling. 

19—2 


292  CLINICAL   PATHOLOGY. 

A  rose-pink  colour  forms  on  the  dish  in  the  presence  of  free 
hydrochloric  acid. 

A  reddish-brown  colour  of  the  residue  after  complete  evapora- 
tion is  due  to  charring  of  the  proteids,  and  must  not  be 
confounded  with  the  rose-pink  of  a  positive  test. 

The  exact  proportions  of  Gunzburg's  reagent  are  phloro- 
glucin,  0*5  gramme  ;  vanillin,  0*25  gramme  ;  absolute 
alcohol,  10  c.c.,  to  which  are  added  10  c.c.  of  the  filtered  test 
meal.  The  reagent  does  not  keep  in  solution  and  must  be 
freshly  prepared.  It  is  not  necessary  to  measure  the  ingredients, 
nor  to  use  such  large  quantities. 

(b)  Estimation    of    the    acidity. — Wash    out    a    clean 

N 
burette    with    a    few    cubic    centimetres    of  —    NaOH    and 

then  fill  with  ~  NaOH. 

Wash  out  a  clean  10  c.c.  pipette  with  a  few  cubic  centimetres 
of  the  test  meal  filtrate,  and  then  measure  10  c.c.  of  the  filtrate 
into  a  large  clean  beaker. 

Dilute  the  filtrate  with  about  4  times  its  volume  of 
distilled  water. 

Add  2  drops  of  Topfer's  solution. 

In  the  presence  of  free  HC1  the  mixture  turns  red. 

Read  the  level  of  fluid  in  the  burette. 

N 
Carefully  run  in  — -  NaOH   until  the    red  colour  becomes 

yellow. 

The  end-point  is  often  blurred,  and  the  change  of  colour 
passes  gradually  from  red,  through  an  orange  colour,  to  a 
lemon -yellow  tint.  As  soon  as  the  last  trace  of  pink  has  gone 
and  the  fluid  is  definitely  yellow,  take  the  reading  of  the  burette. 

Add  2  drops  of  phenol-phthalein. 

N  .  . 

Continue  to  run  in  —  NaOH  until  a  faint,  permanent  pink 

colour  is  produced. 

Again  read  the  burette. 

The  result  is  calculated  as  follows  : — 

The  difference  between  the  original  level  of  the  soda  solution 

and  the  first  reading  gives  the  number  of  cubic  centimetres  of 

N 

-.-pr-NaOH  required  to  neutralise  the  filtrate  to  Topfer's  solu- 


THE   MOUTH -THE    STOMACH.  293 

tion.  In  the  presence  of  a  positive  Giinzburg's  reaction  this 
amount  may  be  taken,  with  certain  reservations,  as  the  equiva- 
lent of  the  free  HC1  present  in  10  c.c.  of  the  test  meal.  The 
calculation  may  be  made  in  terms  of  HC1 ;  thus  if  5  c.c.  of 
decinormal  soda  were  required  for  10  c.c.  of  the  nitrate,  50  c.c. 
would  be  required  for  100  c.c.  of  nitrate  :  1  c.c.  of  decinormal 
soda  is  the  equivalent  of  1  c.c.  of  decinormal  HC1.*  The 
molecular  weight  of  HC1  is  36-5  grammes,  therefore  1  c.c.  of 

-^  HC1  contains  -00365  gramme  of  HC1,  and  50  c.c.  contains 

50  X  '00365  =  0-1825  gramme,  which  is  the  amount  con- 
tained in  100  c.c.  of  the  test  meal.  The  difference  between 
the  original  level  of  the  soda  solution  and  the  second  reading 

N 
gives  the  number  of  cubic  centimetres  of  —  NaOH  required  to 

neutralise  the  filtrate  to  phenol-phthalein.     This  amount  is 

known  as  as  the  "  Total  Acidity,"  and  includes  the  acidity  due 

to  free  HC1,  to  protein  HC1,  to  any  organic  acids  which  may 

be  present,  and  to  acid  salts.    The  total  acidity  is  not  reckoned 

in  terms  of  HC1,  but  is  commonly  given  as  the  number  of 

N 
cubic  centimetres  of  ^  NaOH  required  to  neutralise  100  c.c. 

of  the  filtered  test  meal.  Thus,  if  7  c.c.  of  soda  were  used  for 
10  c.c.  of  the  nitrate,  the  total  acidity  is  recorded  as  70. 

The  fallacies  of  the  estimation.  Presence  of  free 
HC1. — For  reasons  that  need  not  be  entered  into  here,  there 
is  little  doubt  that  free  HC1  may  be  present  in  exceptional 
test  meals,  and  yet  Giinzburg's  test  may  be  negative. 

Topfer's  reagent  may  give  a  red  colour  with  substances 
other  than  free  hydrochloric  acid. 

There  is  no  certain  chemical  test  in  ordinary  use  for  the 
demonstration  of  free  HC1  in  a  test  meal. 

If  Giinzburg's  test  is  positive  free  HC1  is  certainly  present ; 
if  the  test  is  negative  it  is  advisable  to  presume  that  free 
HC1  is  absent.  In  a  normal  test  meal  Giinzburg's  test  is 
positive. 

Amount  of  free  HC1.  —  With  the  exception  of  the 
estimation  of  the  inversion  of  cane  sugar  by  the  polariscope, 
there   is   no   accurate  method  for  the  determination  of  the 

*  A  normal  solution  of  a  monobasic  acid  or  alkali  contains  the  molecular 
weight  of  the  substance  per  litre. 


294  CLINICAL   PATHOLOGY. 

amount  of  HC1  in  a  test  meal.  The  polariscope  method  is 
too  difficult  for  use  as  a  routine  clinical  method,  and  is  not 
described  here. 

Willcox  lays  stress  on  the  amount  of  "  active "  HC1  as 
estimated  by  the  Volhard  process.  By  "  active  "  or  available 
HC1  he  means  the  sum  of  free  HC1  and  HC1  combined  with 
protein.  To  arrive  at  this  measurement,  the  total  chlorides 
present  in  10  c.c.  of  the  test  meal  are  estimated  as  described 
under  the  estimation  of  chlorides  in  the  urine  (page  273).  A 
second  10  c.c.  are  evaporated  to  dryness  and  then  heated  over 
gauze  to  drive  off  the  free  and  protein  HC1 ;  the  residue  is 
dissolved  in  water,  and  the  remaining  or  inorganic  chlorides 
are  similarly  estimated.  The  difference  between  the  total 
and  the  inorganic  chlorides  gives  the  "active"  HC1.  The 
result  almost  invariably  corresponds  closely  with  the  total 
acidity,  and  appears  to  give  a  figure  for  the  "active"  HC1 
considerably  in  excess  of  what  is  actually  present. 

The  Topfer  acidity,  as  described  above,  is  a  roughly  accurate 
measure  of  free  HC1  under  certain  limitations.  The  reagent 
gives  a  red  colour  with  lactic  acid  in  excess,  but  not  in  small 
amount,  as  well  as  with  other  substances  which  may  occa- 
sionally be  present.  It  does  not  react  with  protein  HC1  nor 
with  acid  salts.  Briefly,  when  the  free  HC1  is  in  normal 
amount  or  in  excess  lactic  acid  is  practically  absent,  and  the 
Topfer  acidity  is  a  fairly  accurate  estimation  of  free  HC1.  If 
the  HC1  is  diminished  or  absent,  the  Topfer  acidity  may  record 
considerably  more  free  HC1  than  is  present.  A  definite  Topfer 
acidity  may  be  present  with  a  negative  Giinzburg  reaction. 

For  clinical  purposes  we  require  to  know  if  the  free  HC1  is 
normal,  increased,  decreased,  or  absent.  The  Topfer  acidity 
read  in  conjunction  with  Gunzburg's  test  gives  us  this 
information  in  almost  every  instance. 

The  normal  Topfer  acidity  is  the  equivalent  of  0*08  to  0"1 
gramme  of  HC1  per  cent. 

The  total  acidity. — The  phenol-phthalein  method  is 
accurate,  but  includes  a  variety  of  substances.  Alterations 
in  the  amount  of  the  total  acidity  are  of  considerable  clinical 
significance,  and  in  the  reading  of  a  test  meal  result  chief 
reliance  is  to  be  placed  upon  Gunzburg's  test  and  the  measure 
of  the  total  acidity. 

The  normal  total  acidity  is  from  40  to  50. 


THE    MOUTH— THE    STOMACH.  295 

Summary  of  procedure. — Perforin  Gunzburg's  test  for 

free  HC1. 

If  positive,  add  a  few  drops  of  Topfer's  reagent  to  10  c.c.  of 

N 
nitrate.     Run  in  r-r  NaOH  till  the  red  colour  becomes  yellow. 

Calculate  result  in  terms  of  HC1  (normal   0*08  to  O'l  per 

cent.). 

Add  a  few  drops  of  phenol-phthalein,  and  continue  to  run 

.      N 

in  r-r  NaOH  till  the  colour  becomes  red  again.    Calculate  total 

N 
acidity  in  terms  of  cubic  centimetres  of  r-pr  NaOH  (normal  40 
J  10 

to  50). 

If  Gunzburg's  test  was  negative,  the  Topfer  estimation 
may  be  omitted.  The  whole  procedure  takes  about  10 
minutes. 

The  variations  in  disease. — Free  HC1  may  be  absent, 
as  shown  by  a  negative  Giinzburg  reaction,  in  the  following 
conditions. 

Carcinoma  of  the  stomach  is  almost  invariably  associated  with 
anegativeGiinzburgreaction.  The  free  HC1  may  be  absent  within 
a  very  few  weeks  or  even  a  few  days  of  the  onset  of  symptoms. 
It  is  unfortunately  the  fact  that  an  inoperable  carcinoma  may 
be  found  in  an  equally  short  period.  In  the  rare  form  of 
carcinoma  which  appears  in  a  patient  who  gives  a  long  and 
definite  history  of  previous  "attacks"  of  gastric  ulcer,  free 
HC1  may  persist.  A  positive  Giinzburg  reaction  in  a  patient 
with  a  comparatively  short  history  of  carcinoma  is  very  strong 
evidence  against  a  diagnosis  of  gastric  carcinoma.  Carcinoma 
elsewhere  than  in  the  stomach  may  in  a  minority  of  cases, 
the  majority  of  which  are  cachectic,  lead  to  an  absence  of  HC1. 
The  total  acidity  is  practically  always  low  in  gastric  carcinoma. 
The  average  total  acidity  is  26. 

In  severe  anaemias,  including  pernicious  anaemia,  the  gastric 
juice  has  a  very  low  total  acidity  and  no  free  HC1.  Such 
conditions  are  benefited  by  gastric  treatment  as  an  adjuvant 
to  treatment  directed  against  the  anaemia. 

In  the  rare  condition  known  as  achylia  gastrica,  the  gastric 
juice  is  practically  absent.  Gunzburg's  test  is  negative,  the 
total  acidity  is  in  the  neighbourhood  of  10,  and  pepsin  is 
absent.     In    such    cases   the   passage   of   food    through    the 


296  CLINICAL   PATHOLOGY. 

stomach  is  very  rapid,  the  amount  of  the  test  meal  recovered 
from  the  stomach  is  small,  and  the  meal  may  have  to  be 
withdrawn  in  half  an  hour  or  less. 

In  cholelithiasis  free  HC1  is  almost  always  absent,  an 
important  diagnostic  point  between  gall  stones  and  duodenal 
ulcer. 

In  chronic  gastritis,  particularly  of  the  atonic  type,  and 
in  the  later  stages  of  alcoholic  gastritis,  free  HC1  may  be 
absent.  These  varieties  of  dyspepsia  are  rapidly  relieved 
by  the  administration  of  pepsin  and  hydrochloric  acid  by 
the  mouth. 

Free  HC1  is  almost  invariably  absent  after  a  successful 
gastrojejunostomy  has  been  performed,  apparently  because 
of  the  neutralisation  of  the  gastric  juice  by  admixture  with 
the  biliary  secretion.  This  neutralisation  is  a  most  impor- 
tant and  not  too  widely  recognised  effect  of  the  operation 
for  the  cure  of  duodenal  ulcer.  In  two  such  cases  in  which 
free  HC1  persisted  after  the  operation  peptic  jejunal  ulcers 
followed. 

Free  HC1  may  be  increased  in  the  following  conditions. 

Duodenal  ulcer  is  almost  invariably  accompanied  by  a 
great  increase  in  the  free  acid  and  in  the  total  acidity.  The 
average  free  acidity  in  a  series  of  cases  was  0*17,  and  the 
average  total  acidity  was  69.  Exceptionally  the  total  acidity 
may  rise  above  100.  So  long  as  the  gastric  contents  are  kept 
neutralised  by  drug  treatment  in  these  cases  the  symptoms 
disappear  and  complications  are  most  unlikely  to  occur. 
Simple  gastric  ulcer  is  accompanied  by  a  similar  but  less  marked 
increase  in  the  acidity.  The  average  of  the  free  HC1  is  0'13, 
and  of  the  total  acidity  58.  Higher  readings  are,  of  course, 
frequent.  The  acidity  commonly  falls  very  low,  and  the  free 
HC1  may  disappear,  after  a  considerable  hsematernesis.  The 
fall  in  acidity  is  accompanied  by  an  improvement  in 
symptoms. 

Hyperchlorhydria  or  increase  in  the  acidity  occurs  in  the 
absence  of  ulceration,  and  may  be  very  pronounced.  It 
gives  rise  to  symptoms  which  are  readily  alleviated  by  the 
administration  of  alkalies. 

Increase  in  the  acidity  is  not  uncommon  in  hysterical 
subjects  and  may  be  present  in  some  forms  of  acute  gastritis, 
particularly  of  the  alcoholic  type. 


THE   MOUTH— THE    STOMACH.  297 

(c)  Further  investigations  of  the  test  meal. 

Microscopic  investigation  has  been  already  referred  to. 
It  is  remarkable  how  rarely  anything  of  practical  importance 
is  made  out  by  this  means. 

Lactic  acid,  if  present  in  sufficient  amount  to  give  the 
ordinary  tests,  is  abnormal.  The  presence  of  lactic  acid  is 
practically  always  associated  with  a  negative  Giinzburg 
reaction  and  a  total  acidity  of  moderate  amount,  that  is  from 
30  to  40.  The  demonstration  of  lactic  acid  in  a  test  meal  is 
no  real  additional  evidence  of  carcinoma.  Lactic  acid  is 
commonly  present  in  carcinoma  cases,  as  well  as  in  chronic 
gastritis  with  diminution  or  absence  of  HC1. 

To  test  for  lactic  acid  proceed  as  follows  :  — 

To  10  c.c.  of  the  filtered  test  meal  add  about  20  c.c.  of 
ether. 

Mix  thoroughly  by  repeated  inversion  in  a  separating  funnel. 

Allow  to  stand  for  half  an  hour. 

Evaporate  down  the  ether  extract  in  a  beaker  standing 
in  hot  water  (not  over  a  flame). 

To  the  residue  add  about  3  c.c.  of  distilled  water. 

With  the  watery  solution  perform  Uffelmann's  test  as 
follows  : — 

Fill  a  test  tube  one-third  full  with  1  in  20  carbolic 
acid. 

Add  1  drop  of  ferric  chloride. 

Dilute  the  mixture  with  water  until  the  amethyst  blue 
colour  is  just  transparent. 

Add  the  watery  solution  of  the  ether  residue  to  the  mixture. 

In  the  presence  of  lactic  acid  the  amethyst  blue  of  the 
reagent  becomes  decolorised  and  changed  to  a  canary  yellow 
colour. 

The  test  is  distinctive,  but  not  very  delicate. 

Pepsin. — The  pepsin  content  of  a  test  meal  is  of  con- 
siderable importance  and  is  readily  estimated.  Pepsin 
appears  to  vary  roughl  ywith  the  amount  of  the  free  HC1  and 
the  total  acidity.  It  is,  however,  rarely  absent  except  in  cases 
of  achylia  gastrica. 

The  edestin  method  for  the  estimation  of  pepsin  is  a  simple 
and  very  reliable  one. 

Edestin  is  a  crystalline  albumin  obtained  from  eggs.  It  is 
converted  by  pepsin  into  edeston. 


298  CLINICAL     PATHOLOGY. 

Edeston  is  soluble  in  sodium  chloride.  Edestin  is  pre- 
cipitated by  sodium  chloride. 

Pure  edestin  can  be  obtained  from  Merck. 
To  perform  the  estimation  : — 
Get  ready  the  following  solutions. 

(A)  ^  HC1 30  c.e. 

Distilled  water        ...         70  c.c. 
Edestin  .         .         .         .  0-l  gramme. 

The  edestin  should  pass  readily  into  solution,  but  if  it  does 
not  the  mixture  may  be  heated  and  if  necessary  filtered. 

(B)  A  saturated  solution  of  NaCl. 

(C)  The  filtrate  of  the  test  meal. 

Into  each  of  10  clean  test  tubes  measure  1  c.c.  of  the 
edestin  solution. 

Number  each  test  tube  from  1  to  10. 

To  No.  1  add  1  c.c.  of  the  test  meal.  Mix.  Take  1  c.c.  of 
the  mixture  and  add  to  No.  2.  Mix.  Take  1  c.c.  of  the 
mixture  and  add  to  No.  3,  and  so  on  to  No.  10. 

Leave  at  room  temperature  for  half  an  hour. 

Add  a  few  drops  of  NaCl  solution  to  each  tube.  In  the 
tubes  which  remain  clear  peptic  action  is  complete.  With 
normal  gastric  juice  the  last  clear  tube  will  be  6,  7  or  8. 

Rennin  is  considered  by  some  authorities  to  be  identical 
with  pepsin,  and  is  allowed  by  all  to  vary  in  amount  directly 
with  the  quantity  of  pepsin  present.  The  rennin  can  be 
estimated  by  the  coagulating  action  of  the  gastric  contents  on 
milk. 

To  perform  the  estimation  proceed  as  follows : — 

Label  10  clean  test  tubes  from  1  to  10. 

Into  each  tube  measure  1  c.c.  of  distilled  water. 

Into  tube  1  measure  with  a  pipette  1  c.c.  of  the  test  meal 
filtrate.  Mix.  Take  1  c.c.  of  the  mixture  and  add  to  tube  2. 
Mix  again  and  take  1  c.c.  of  the  mixture  and  add  to  tube  3. 
Continue  the  process  to  tube  10.  The  filtrate  is  thus  pro- 
gressively diluted  by  half  in  each  tube. 

To  each  tube  add  5  c.c.  of  fresh  milk. 

Stand  the  tubes  in  a  crate  in  water  at  room  temperature  for 
2  hours. 

Stand  in  a  water  bath  at  37°  C.  for  5  minutes. 

Remove  and  examine  the  tubes  for  coagulation  of  the  milk. 


THE   MOUTH— THE    STOMACH.  299 

With  normal  gastric  contents  coagulation  will  have  occurred 
up  to  tubes  6,  7  or  8. 

Blood  should  not  be  present  in  a  test  meal,  but  may  be 
found  in  considerable  amount  in  cases  of  carcinomatous, 
and  less  often  of  simple  gastric  ulcer.  Blood  in  quantity 
visible  to  the  naked  eye  may  be  confirmed  by  microscopic 
examination  ;  blood  in  less  amount  should  be  tested  for  as 
follows  : — 

To  5  c.c.  of  filtered  test  meal  in  a  test  tube  add  2  c.c.  of 
acetic  acid. 

Shake  with  an  excess  of  ether. 

Allow  to  stand. 

Pour  off  clear  ether. 

Add  to  the  ether  10  drops  of  a  freshly- prepared  alcoholic 
solution  of  guaiacum  resin  and  20  drops  of  a  5  per  cent, 
solution  of  hydrogen  peroxide. 

In  the  presence  of  blood  the  mixture  becomes  coloured  blue 
of  greater  or  less  intensity. 

Bile  may  be  present  in  the  test  meal  in  cases  of  jaundice, 
and  after  the  performance  of  a  gastrojejunostomy.  The 
bile  pigments  should  be  tested  for  in  the  usual  way. 

Other  gastric  investigations. — (1)  The  motility  of  the 
stomach. 

Sahli's  desmoid  test. — The  test  does  not  involve  the  use 
of  a  stomach-tube  and  depends  upon  the  fact  that  gastric  juice 
alone  can  digest  raw  catgut,  which  is  quite  insoluble  in  the 
pancreatic  juice.  The  test  is  not  precisely  one  of  the  motility 
of  the  stomach,  but  a  general  test  of  the  gastric  digestive 
function. 

The  test  is  performed  as  follows : — 

A  rubber  membrane  of  the  finest  Para  rubber,  0*2  mm. 
thick,  is  used  as  a  wrapper.  In  it  is  placed  a  methylene  blue 
pill  made  according  to  the  following  prescription  : — 

Methylene  blue  (medicinal)  .         .        10"0  grammes. 
Pulv.  glycyrrhizae  .         .         .        10"0         „ 

Bismuth  subnitr.  .         .         .       100*0         „ 

Glucose  syrup  (sat.)  q.s. 

Divide  into  200  pills. 

The  edges  of  the  membrane  are  closed  firmly,  but  gently,  by 
winding  round  them  a  piece  of  the  best  catgut  0*3  mm.  thick. 

During  the  ordinary  mid-day  meal,  and  immediately  after 


300  CLINICAL  PATHOLOGY. 

soup,  the  patient  is  instructed  to  swallow  the  sac  with  the  aid 
of  a  little  water.  The  patient  is  told  to  pass  water  at  ,5  p.m., 
at  7  p.m.,  and  on  rising  the  following  morning.  The  samples 
of  urine  are  kept  separate.  If  the  methylene  blue  appears 
in  the  urine  of  the  same  evening  or  of  the  following  morning 
digestion  is  considered  to  be  normal.  If  the  methylene  blue 
fails  to  appear,  either  gastric  digestion  is  incomplete,  or  the 
stomach  is  hypermotile.  A  delayed  reaction  taking  place  on 
the  afternoon  or  evening  of  the  following  day  points  to  incom- 
plete digestion  in  the  stomach  associated  with  hypoacidity  and 
gastric  stasis. 

The  iodopin  test. — This  test  is  considered  to  bear  directly 
upon  the  motility  of  the  stomach. 

It  is  performed  as  follows  : — 

A  quarter  of  an  hour  after  a  test  breakfast  the  patient  is 
given  a  teaspoonful  of  iodopin.  Subsequently  the  saliva  is 
tested  every  15  minutes  for  iodine.  The  patient  is  provided 
with  a  series  of  papers  which  have  been  soaked  in  a  starch 
emulsion  and  instructed  to  salivate  upon  them  at  regular 
intervals.  Normally  the  iodine  appears  in  the  saliva  in  from 
60  to  75  minutes. 

It  cannot  be  said  that  either  of  the  above  tests  are 
particularly  satisfactory.  Nor  can  the  information  derived 
from  them  compare  with  the  results  of  an  accurate  investiga- 
tion of  a  test  meal.  Both  methods,  however,  as  well  as 
numerous  others  of  a  similar  kind,  are  fairly  extensively  used. 

The  fasting  stomach. — The  motility  of  the  stomach 
can  be  estimated  by  examination  in  the  fasting  condition. 
The  ordinary  evening  meal  is  taken  and  no  other  food  until 
10  o'clock  on  the  following  morning,  when  a  tube  is  passed 
and  the  contents,  if  any,  are  removed.  Under  normal 
conditions  the  stomach  should  be  quite  empty  within  7  or 
8  hours  of  an  abundant  meal.  If  particles  of  food  are 
found  the  next  morning  motor  insufficiency  is  present.  If 
a  considerable  quantity  of  fluid  is  withdrawn  containing 
a  definite  percentage  of  hydrochloric  acid  and  no  particles 
of  food,  hyperacidity  is  present.  A  little  fluid  containing  a 
faint  trace  of  acid  is  not  abnormal,  since  it  may  be  induced  by 
the  passage  of  the  tube.  The  test  for  motility  of  the  stomach 
may  be  combined  with  the  examination  of  the  test  breakfast. 
The  ordinary  dinner  is  given  in  the  evening,  and  with  the 


THE    MOUTH— THE    STOMACH.  301 

dinner  half  a  dozen  or  more  raisins  are  swallowed  whole.  At 
9  o'clock  the  following  morning  the  test  breakfast  is  given. 
At  10  o'clock  the  breakfast  is  withdrawn  and  examined  in  the 
ordinary  way.  If  the  raisins  or  fragments  of  them  are 
present  in  the  gastric  contents,  hypomotility  can  be  diagnosed. 

(2)  Examination  of  the  vomit. — The  points  to  be 
investigated  in  vomited  material — without  considering  the 
detection  of  poisons — are  the  following  : — 

Blood  should  be  examined  for  and,  unless  in  very  obvious 
amount,  tested  for  by  the  method  given  above.  Among  the 
more  important  conditions  associated  with  hsemateniesis  are 
cirrhosis  of  the  liver,  gastric  ulcer,  simple  and  malignant, 
and  rarely  duodenal  ulcer.  Small  quantities  of  blood  in  the 
vomit  are  of  little  importance,  and  may  be  produced  by  the 
act  of  vomiting  from  a  tiny  ruptured  vessel.  Larger  amounts 
may  come  from  elsewhere,  as  from  the  nose,  or  lungs,  or 
mouth,  and  appear  after  being  swallowed.  Profuse  haemor- 
rhage from  the  gastric  mucous  membrane  may  follow  minute 
erosions  in  the  absence  of  macroscopic  ulceration,  and  is 
usually  accompanied  by  hyperacidity. 

Bile  is  of  little  importance,  and  is  likely  to  occur  with  any 
form  of  vomiting  if  prolonged. 

Faecal  vomiting  is  characteristic  of  intestinal  obstruc- 
tion. The  vomit  is  of  a  brownish-black  colour  resembling 
that  of  altered  blood,  and  of  a  faecal  odour.  Faecal 
vomiting  is  very  occasionally  met  with  in  purely  functional 
cases,  and  it  may  happen  with  such  patients  that  the  odour 
and  colour  of  a  turpentine  or  soap  enema  may  appear  in 
the  vomit. 

Mucus  in  large,  tough  masses  may  be  seen  in  the  vomit 
in  cases  of  chronic  catarrhal  gastritis  with  hypoacidity.  It 
is  found  in  the  morning  vomit  of  chronic  alcoholic  patients. 

The  acidity  of  the  vomit  is  in  the  majority  of  cases 
scarcely  worth  estimating  ;  but  the  expulsion  of  a  quantity 
of  acid  fluid  some  hours  after  a  meal  is  evidence  of  hyper- 
secretion. 

(3)  The  bacteriology  of  the  stomach.— Although  it  is 
held  by  some  authorities  that  many  gastric  disorders  are  due 
to  bacterial  infection,  the  bacteriological  investigation  of  the 
stomach  contents  is  at  the  present  of  little  value.  The  anti- 
septic action  of  the  gastric  juice  is  no  doubt  slighter  than  was 


302  CLINICAL   PATHOLOGY. 

formerly  supposed,  and  micro-organisms  of  any  and  every 
kind  can,  on  occasion,  be  found  even  in  highly  acid  gastric 
contents.  The  presence  of  the  Oppler-Boas  bacillus  in  the 
test  meal  or  the  vomit  is  still  held  by  some  to  be  characteristic 
of  gastric  carcinoma.  The  bacillus  is  long,  thin,  and  may 
occur  in  chains.  It  is  often  associated  with  sarcinse,  and 
is  not  frequently  found  in  cases  of  gastro-stasis  with 
hypoacidity. 

Among  the  methods  recommended  for  examination  of  the 
bacterial  content  of  the  stomach  is  the  washing  of  the  fasting 
stomach.  A  sterile  tube  is  passed,  and  the  stomach  is  washed 
out  into  a  bowl  with  sterile  water.  The  water  is  examined 
microscopically  and  culturally.  As  might  be  expected,  it  is 
quite  unusual  to  obtain  a  sterile  fluid  by  this  method.  The 
organisms  found  may  come  from  the  stomach,  from  the 
oesophagus  or  mouth  by  contamination  of  the  end  of  the  tube, 
or  from  the  swallowed  saliva. 

The  organisms  most  commonly  obtained  are  streptococci, 
staphylococci,  bacilli  of  the  coli  or  proteus  group,  and  sarcinse. 
The  significance  to  be  attached  to  any  of  them  is  probably 
slight. 


CHAPTER  XXI. 

the  pancreas — the  liver — the  spleen — the  peritoneum. 
The  Pancreas. 

There  are  no  satisfactory  clinical  methods  for  investigating 
the  functions  of  the  pancreas  and  the  efficiency  of  the 
pancreatic  juice. 

The  pancreatic  reaction  of  Cammidge  has  been  described  in 
the  section  on  the  urine.  It  is  of  very  doubtful  clinical  value, 
and  recently  further  experimental  evidence  has  been  adduced 
against  it.  It  is  stated  that  in  test  C  the  glycuronic  acid  is 
incompletely  removed,  and  that  the  Cammidge  crystals  are 
derived  from  glycuronic  acid,  which  is  known  to  occur  in  the 
urine  in  variable  amount  under  very  varying  circumstances 
such  as  are  not  necessarily  connected  with  pancreatic  disease. 
The  investigation  of  the  pancreatic  activity  in  fat  metabolism 
will  be  described  in  the  chapter  on  the  faeces. 

Pancreatic  efficiency  is  also  investigated  by  a  method  similar 
to  the  "  desmoid "  test  of  Sahli  for  gastric  efficiency.  The 
test  is  performed  by  enclosing  a  little  salol  in  a  gelatin 
capsule  hardened  with  formalin.  The  capsule  is  given  with 
the  ordinary  morning  breakfast  or  with  the  test  breakfast. 
The  gastric  juice  is  unable  to  dissolve  the  capsule,  which 
passes  unchanged  through  the  stomach,  but  is  readily 
digested  by  the  pancreatic  juice.  The  salol  is  then  tested  for 
in  the  urine  in  samples  taken  after  2,  3,  4,  5,  and  6  hours. 
The  test  for  salol  in  the  urine  is  to  add  a  few  drops  of 
perchloride  of  iron,  which  gives  an  amethyst  blue  colour  in 
the  presence  of  salol.  In  cases  of  pancreatic  insufficiency 
the  appearance  of  the  salol  in  the  urine  is  much  delayed  or 
may  not  occur.  In  cases  of  diarrhoea  with  a  normal 
pancreatic  digestion  the  salol  reaction  appears  early.  In 
health  the  salol  appears  in  the  urine  in  about  5  hours. 

Pancreatic  cysts,  and  the  methods  of  testing  ferments  in 
them,  have  been  mentioned  in  the  section  on  pancreatic 
fluids. 


304  CLINICAL   PATHOLOGY. 

The  Liver. 

The  test  for  new  growths  of  the  liver,  or  abscess  of  the 
liver,  by  demonstration  of  creatine  in  the  urine  has  been 
already  described.  In  acute  yellow  atrophy  of  the  liver,  and 
in  phosphorus  poisoning,  crystals  of  leucine  and  tyrosine  are 
to  be  looked  for  in  the  urine.  In  cases  of  biliary  obstruction 
the  patient  is,  as  a  rule,  obviously  jaundiced  and  the  condition 
may  be  confirmed  by  the  demonstration  of  bile  pigment  in  the 
serum.  The  pigments  are  also  present  in  the  urine,  but 
absent  from  the  stools.  The  serum  in  such  cases  is  often 
markedly  bile-tinged  at  a  period  when  clinical  evidence  of 
jaundice  is  lacking.  Puncture  of  the  liver  is  occasionally 
performed  in  cases  of  suspected  liver  abscess  or  of  hydatid 
cyst.  Except  during  the  course  of  an  operation,  puncture 
of  the  liver  should  be  performed  cautiously  and  only  in  the 
area  of  an  evidently  superficial  tumour.  A  fine  hypodermic 
needle  should  be  used,  The  fluid  withdrawn  from  a  tropical 
abscess  is  very  typical  in  appearance,  having  a  considerable 
resemblance  to  anchovy  sauce.  Unless  a  secondary  infection 
has  occurred,  the  fluid  is  odourless.  Amoebae  should  be 
looked  for  in  fresh  preparations  of  the  fluid,  but  are  rarely 
detected,  since  they  seem  to  be  mainly  confined  to  the  lining 
wall  of  the  abscess.  After  the  abscess  has  been  opened  and 
drained,  the  amoebae  are  likely  to  appear  on  the  second  or 
third  day.  The  methods  of  detecting  amoebae  in  the  stools 
should  be  applied  in  the  examination  of  "  pus  "  from  the  liver. 
Dried  films  made  from  the  pus  show  little  besides  necrotic 
cells  and  amorphous  debris.  Cultures  are  almost  invariably 
sterile. 

The  nature  of  the  fluid  of  a  hydatid  cyst  has  been  described 
in  the  section  on  puncture  fluids.  The  characteristic  hydatid 
hooklets  should  always  be  sought  for. 

Bile  is  occasionally  obtained  by  puncture  through  the 
abdominal  wall  of  a  distended  gall  bladder ;  it  is  more 
frequently  and  justifiably  received  in  the  laboratory  from 
the  operating  theatre.  In  cases  of  distension  of  the  gall 
bladder  due  to  a  calculus  impacted  in  the  cystic  duct,  the  fluid 
is  often  colourless  or  merely  tinged  with  bile  pigment ; 
proteids  are  scanty,  and  mucin  is  usually  present  in  consider- 
able amount.     If  no  distension  of  the  gall  bladder  is  present, 


PANCREAS— LIVER— SPLEEN— PERITONEUM.     305 

the  normal  bile  is  greenish  in  colour  and  of  a  glycerine-like 
consistency.  When  inflammation  is  present,  or  gall-stones 
are  found  in  addition,  the  bile  may  be  either  paler  or  darker 
in  colour,  and  is  almost  always  more  syrupy  and  ropy  in 
consistence.  More  rarely  the  gall  bladder  may  be  filled  with 
pus,  and  actual  gangrene  of  the  walls  may  be  present.  The 
association  of  gall-stone  formation  with  microbic  infection 
of  the  bile  passages  is  undoubted.  The  path  by  which  the 
organisms  reach  the  gall  bladder  is  in  dispute,  but  the 
character  of  the  bacteria  commonly  found,  and  the  direct 
communication  between  duodenum  and  gall  bladder  would 
point  to  an  intestinal  infection  as  the  most  obvious  route. 

The  bile  in  such  cases  should  be  examined  by  cytological 
and  cultural  methods.  Gall-stones,  if  present,  should  be 
culturally  examined  and  their  composition  investigated. 

In  about  20  per  cent,  of  cases  with  gall-stones  the  bladder 
is  found  to  be  sterile,  and  formerly  considerable  importance 
was  attached  to  the  antiseptic  properties  of  the  bile  in 
preventing  the  spread  of  organisms  from  the  alimentary 
tract. 

It  is  probable  that  the  antiseptic  action  of  the  bile  salts 
is  of  importance,  but  it  appears  to  have  little  effect  upon 
organisms  of  the  colon  and  typhoid  groups.  This  action  has 
been  taken  advantage  of  in  the  preparation  of  MacConkey's 
bile  salt  medium.  The  growth  of  cocci  is  completely  inhibited 
by  the  presence  of  the  sodium  taurocholate  in  the  medium, 
while  the  colon  and  typhoid  bacilli  grow  abundantly.  The 
antiseptic  action  of  the  bile  is  possibly  diminished  under 
various  pathological  conditions,  and  organisms  other  than 
those  of  the  coli-typhoid  group  may  be  found. 

The  following  are  among  the  organisms  most  commonly 
met  with  in  the  gall  bladder  : — 

The  bacillus  coli  is  the  organism  most  frequently  present 
in  inflammatory  conditions  of  the  gall  bladder,  either  alone 
or  in  association  with  gall-stones.  There  is  no  difficulty  in 
recovering  and  identifying  it  in  cultures  made  from  the  bile 
by  the  ordinary  methods. 

The  typhoid  bacillus  may  be  present  in  the  gall  bladder 
many  years  after  an  attack  of  typhoid  fever.  The  association 
of  typhoid  fever  with  the  subsequent  production  of  gall-stones 
is  well  known.     The  detection  of  typhoid  bacilli  in  the  gall 

p.  20 


306  CLINICAL   PATHOLOGY. 

bladder  is  of  great  importance  apart  from  the  production  of 
calculi,  since  the  latency  of  the  organisms  in  this  situation 
is  probably  responsible  for  the  majority  of  instances  of 
"  typhoid  carriers."  The  organisms  readily  pass  down  the 
intestinal  tract  and  are  excreted  in  the  faeces.  Such  a 
typhoid  carrier  occupying  the  position  of  a  cook  or  a  domestic 
servant  is  a  serious  menace  to  the  community  with  which  he  or 
she  is  associated.  The  detection  of  typhoid  bacilli  in  the  bile 
is  a  simple  matter,  if  the  bile  is  available  during  an  opera- 
tion for  gall-stones.  Typhoid  carriers  can  otherwise  be 
recognised  by  the  detection  of  the  bacilli  in  the  stools  and 
by  examination  of  the  serum  for  the  presence  of  typhoid 
agglutinins. 

Streptococci,  staphylococci,  and  much  more  rarely 
pneumococci,  are  among  other  organisms  which  may  be 
obtained  in  cultures  from  the  bile. 

Gall-stones  may  be  either  single  or  multiple ;  if  multiple 
they  are  usually  facetted.  They  may  be  found  in  the  gall 
bladder,  cystic  duct  or  common  duct,  or  they  may  have 
escaped  into  the  intestine.  Barely  they  may  have  ulcerated 
through  into  the  peritoneal  cavity  or  through  the  anterior 
abdominal  wall.  Exceptionally  small  calculi  may  be  found 
in  the  finer  hepatic  ducts  in  the  substance  of  the  liver. 
The  great  majority  of  gall-stones  consist  mainly  or  entirely 
of  cholesterin.  Pure  cholesterin  calculi  are  of  a  translucent 
yellow  colour  and  very  light.  The  stones  not  infrequently  con- 
tain in  addition  to  the  cholesterin  varying  amounts  of  biliary 
pigment,  either  bilirubin  or  biliverdin.  More  rarely  small 
calculi  are  found  consisting  entirely  of  biliverdin  or  bilirubin 
with  a  small  admixture  of  calcium.  Calcium  carbonate  and 
phosphate  calculi  are  common  in  some  animals,  but  very  rare 
in  man. 

The  following  tests  for  cholesterin  should  be  applied  to  a 
gall-stone  after  powdering  a  portion  of  it. 

(1)  Dissolve  some  of  the  powder  in  ether. 

Evaporate  down  the  ether  extract  in  hot  water  (not  over 

a  flame). 
Dissolve  a  small  portion  of  the  residue  in  a  little  absolute 

alcohol. 
Allow  a  few  drops  of  the  alcoholic  extract  to  evaporate 
at  room  temperature  on  a  microscope  slide. 


PANCREAS— LIVER— SPLEEN— PERITONEUM.     807 


Examine  the  slide  for  the  typical  flat  crystals  of  choles- 

terin. 
The  crystals  consist  of  rhombic  plates  notched  at  one 

angle  (Fig.  18). 

(2)  Dissolve  another  portion  of  the  ether  residue  in  pure 

anhydrous  ether. 
Allow  a  few  drops  to  evaporate  on  a  slide  as  above. 
Cholesterin  crystallises  from  ether  in  the  form  of  fine 

needles. 

(3)  Dissolve   another   portion    of    the    ether   residue   in    a 

small  quantity  of 
chloroform    in    a 
test  tube. 
Add     an    equal 
volume  of  concen- 
trated   sulphuric 
acid  and  mix. 
The  chloroform  be- 
comes coloured  a 
deep  red  and  rises 
to  the  surface  of 
the  mixture. 
The  sulphuric  acid 
shows  a  brilliant 
green    fluores- 
cence. 
This  test  is  known  as  Salkowski's  reaction. 
Bile  pigments  in  gall-stones  are  recognised  by  their  colour 
and  by  their  reaction  to  Gmelin's  test. 

The  tests  for  carbonates,  phosphates,  and  calcium  are 
conducted  in  the  same  manner  as  described  under  the  analysis 
of  urinary  calculi. 

The  bacteriological  investigation  of  gall-stones  is  conducted 
as  follows : — 

If  the  stone  has  been  removed  by  the  surgeon  from  the 
bile  passages  and  placed  direct  in  a  sterile  receptacle 
no  preliminary  treatment  is  necessary.  If,  however,  the 
surface  of  the  stone  has  become  contaminated  it  should  be 
placed  on  a  clean  dish  and  washed  repeatedly  with  sterile 
water.  The  stone  is  then  placed  in  a  sterile  mortar  and 
crushed.     Culture  tubes  are  inoculated  with  portions  of  the 

20—2 


Fig.  18. — Cholesterin  Crystals. 


308  CLINICAL   PATHOLOGY. 

crushed  calculus.     The  remainder  can  be  used  for  chemical 
analysis. 

The  bacteria  found  in  gall-stones  are  precisely  similar  to 
those  present  in  the  bile.  A  considerable  percentage  of  gall- 
stones, however,  are  sterile. 

The  Spleen. 

Owing  to  the  remarkable  changes  which  may  take  place 
in  the  spleen  in  certain  blood  diseases,  the  part  which  it 
plays  in  them  is  apt  to  be  over-estimated.  There  is  little 
evidence  that  enlargement  of  the  spleen  in  the  primary 
anaemias  is  anything  but  a  secondary  phenomenon,  or  that 
in  adult  life  the  spleen  takes  any  prominent  or  special  share 
in  blood  formation.  We  are  not  concerned  with  the  various 
theories  advanced  as  to  the  functions  of  the  spleen,  but  it 
may  be  mentioned  that,  apart  from  any  possible  controlling 
action  on  the  iron  metabolism  of  the  body,  almost  the  only 
certain  function  of  the  spleen  is  to  act  as  a  reservoir  for  the 
portal  circulation  during  digestion. 

Tumours  in  the  left  hyphochondrium  continue  to  furnish 
the  clinician  with  interesting  problems  for  diagnosis.  If  such 
a  tumour  be  present  several  pathological  investigations  may 
be  necessary. 

It  is  not  intended  to  imply  that  all  these  investigations 
should  be  performed  in  any  case  of  splenic  enlargement. 
The  clinical  examination  of  the  patient  will  almost  always 
indicate  which  investigation  is  necessary. 

Examination  of  the  cells  of  the  blood.— In  all  cases 
of  splenic  enlargement  some  examination  of  the  blood  must  be 
made,  and  this  in  a  considerable  percentage  of  cases  will 
establish  the  diagnosis. 

In  myeloid  leukaemia,  among  the  primary  blood  diseases,  there 
is  almost  always  a  considerable  enlargement  of  the  spleen,  and 
the  enlargement  is  often  so  great  that  the  spleen  comes  to 
occupy  more  than  half  the  abdominal  cavity. 

In  lymphoid  leukaemia  the  spleen  is  as  a  rule  moderately 
enlarged,  but  occasionally  the  enlargement  is  insufficient  for 
detection  by  clinical  means. 

In  pernicious  anaemia  the  spleen  is  enlarged  and  commonly 
of  such  a  size  as  to  be  just  palpable. 


PANCREAS— LIVER— SPLEEN— PERITONEUM.     309 

Enlargement  of  the  spleen  from  any  of  the  above  three 
causes  is  readily  recognised  by  the  ordinary  examination  of 
the  blood. 

In  chlorosis  there  is  no  evident  enlargement  of  the  spleen, 
and  if  considerable  splenic  enlargement  is  present  with  an 
anaemia  of  the  chlorotic  type  some  other  cause  for  the 
splenomegaly  must  be  sought. 

In  erythremia  a  considerable  increase  in  the  size  of  the 
spleen  is  usual.  Patients  with  this  condition  are,  as  a  rule, 
cyanosed,  and  the  blood  shows  great  excess  in  the  number  of 
red  cells. 

Marked  enlargement  of  the  spleen  in  small  children  may  be 
associated  with  active  rickets  or  with  congenital  syphilis,  and 
with  little  change  in  the  blood ;  or  these  diseases  may  be 
absent  and  the  blood  show  the  pronounced,  if  variable,  changes 
of  the  splenic  anaemia  of  children. 

Occasionally  marked  enlargement  of  the  spleen  in  children 
may  occur  in  the  absence  of  other  obvious  disease  or  of  changes 
in  the  blood. 

In  primary  splenomegaly  or  splenic  anaemia  of  adults,  and 
in  Banti's  disease,  the  size  of  the  spleen  is  usually  very  con- 
siderable, and  may  even  rival  that  found  in  myeloid  leukaemia. 
The  changes  in  the  blood  in  this  condition  are  not  diagnostic, 
but  if  a  considerable  anaemia  of  the  secondary  type  is  absent 
and  there  is  no  leucopenia,  the  diagnosis  of  primary  spleno- 
megaly is  probably  incorrect. 

In  acholuric  family  jaundice  the  size  of  the  spleen  is  con- 
siderable. The  condition  may  be  recognised  by  the  abnormal 
fragility  of  the  red  cells  in  dilute  saline. 

In  acute  inflammatory  conditions  a  slight  enlargement  of 
the  spleen  is  frequently  met  with.  The  blood  shows  an  increase 
in  the  total  leucocytes,  with  a  relative  increase  in  the  polynu- 
clear  neutrophils  and  large  hyalines. 

Examination  of  the  blood  serum. — The  moderate 
enlargement  of  the  spleen  which  occurs  in  typhoid  fever,  and 
the  more  marked  enlargement  present  in  Malta  fever,  may  be 
confirmed  by  the  demonstration  of  the  specific  agglutinins  in 
the  serum. 

Enlargement  of  the  spleen  is  often  considerable  in  congenital 
syphilis,  and  in  the  amyloid  degeneration  which  may  follow 
congenital  or  acquired  syphilis.     The  spleen  may  be  palpable 


310  CLINICAL   PATHOLOGY. 

in  the  secondary  stage  of  syphilis.  A  positive  Wassermann 
reaction  will  be  found  in  the  serum. 

The  parasitology  of  the  blood.— Definite  enlargement 
of  the  spleen  may  follow  infarction  in  the  course  of  infective 
endocarditis,  and  the  nature  of  the  disease  may  be  established 
by  cultivation  of  the  blood.  In  relapsing  fever  the  spirillum 
is  present  in  blood  films.  In  malaria  a  moderate  enlargement 
of  the  spleen  is  usual,  and  the  parasites  can  nearly  always  be 
found  in  film  preparations.  The  enlargement  of  the  spleen 
which  accompanies  malaria  may  exceptionally  persist  for  a  con- 
siderable time  after  the  patient  has  left  the  malarial  district : 
in  such  cases  no  parasites  may  be  found  in  the  blood.  A  very 
considerable  enlargement  of  the  spleen  without  the  typical 
signs  of  malaria  and  in  the  absence  of  malarial  parasites  in  the 
blood  is  probably  due  to  some  other  cause.  A  past  history  of 
malaria  is  not  in  itself  sufficient  to  account  for  a  tumour  in 
the  left  hypochondrium. 

Spleen  puncture  should  be  performed  in  cases  of  splenic 
enlargement  which  may  possibly  be  the  result  of  kala-azar. 
The  puncture  is  without  risk  if  performed  with  a  fine 
hypodermic  needle.  Sufficient  material  can  usually  be  with- 
drawn to  demonstrate  the  Leishman-Donovan  bodies. 

The  urine  must  always  be  carefully  examined  in  all  cases 
of  tumour  in  the  left  hypochondrium.  A  renal  swelling  on 
clinical  examination  may  very  closely  resemble  an  enlarged 
spleen,  and  the  detection  of  pus  in  the  urine  may  serve  to 
clinch  the  diagnosis.  If  the  spleen  is  enlarged  from  amyloid 
disease  the  kidneys  will  probably  also  be  affected,  and  the  urine 
will  contain  albumin,  often  in  considerable  amount. 

Cirrhosis  of  the  liver  is  often  associated  with  a  hard 
and  palpable  spleen.  The  diagnosis  as  a  rule  is  readily  made 
on  clinical  grounds. 

Hodgkin's  disease  is  frequently  associated  with  a  splenic 
enlargement  which  may  be  considerable.  None  of  the  exami- 
nations just  described  are  likely  to  throw  any  light  upon  the 
diagnosis.  A  general  enlargement  of  the  glands,  however,  is 
nearly  always  present,  and  it  is  justifiable  to  remove  a  single 
superficial  gland  for  histological  investigation. 

Tuberculosis,  particularly  of  the  generalised  glandular 
type,  may  lead  to  considerable  splenic  enlargement.  Labora- 
tory investigations,  in  the  absence  of  a  recognised  complement 


PANCREAS— LIVER— SPLEEN— PERITONEUM.     311 

deviation  test  for  tuberculosis,  are  mainly  negative.  The 
diagnosis  is  often  arrived  at  by  a  process  of  exclusion,  and 
commonly  rests  between  tuberculosis  and  Hodgkin's  disease. 

Tumours  of  the  spleen,  whether  primary  or  secondary,  are 
extremely  rare.  Cysts  of  the  spleen  and  gummata  of  the 
spleen  are  likewise  in  the  nature  of  pathological  curiosities. 

Renal  tumours,  tumours  of  the  stomach  or  intestine,  and 
tumours  or  cysts  of  the  omentum,  mesentery,  or  pancreas 
may  all  closely  resemble  splenic  tumours  on  physical 
examination. 

It  is  evident  that  a  very  large  number  of  diseases  may  be 
accompanied  by  splenic  enlargement.  In  the  majority  of 
them,  however,  the  pathological  condition  is  fairly  obvious 
on  clinical  grounds  and  the  enlargement  of  the  spleen  is  of 
secondary  importance.  Particularly  is  this  the  case  in  slight 
enlargement  of  the  spleen  to  palpation.  Great  enlargement 
of  the  spleen  is  rare,  and  the  most  usual  causes  are  myeloid 
leukaemia,  splenic  anaemia,  kala-azar,  the  splenic  anaemia  of 
children,  and  rarely  chronic  lymphoid  leukaemia.  These 
diseases  can  nearly  always  be  recognised  by  a  blood  examina- 
tion or  by  spleen  puncture.  Moderate  enlargements  of  the 
spleen  are  more  difficult  of  diagnosis,  and  the  commonest 
causes  are  tuberculosis,  Hodgkin's  disease,  and  in  children 
congenital  syphilis  and  rickets. 

The  Peeitoneum. 

The  cellular  nature  of  peritoneal  exudates  has  been  sufficiently 
indicated  in  the  section  dealing  with  puncture  fluids.  The 
bacteriological  examination  of  exudates  obtained  at  operation 
may  be  further  considered  in  reference  to  the  abdominal  lesion 
responsible  for  the  inflamed  peritoneum. 

Gastric  ulcer. — Perforation  of  a  gastric  ulcer  may  lead  to 
a  localised  abscess  or  to  a  generalised  peritonitis.  A  number 
of  different  organisms  may  be  found  in  the  exudate,  but  in 
the  majority  of  cases  the  bacterium  which  predominates  in  film 
preparations,  and  often  in  cultures,  is  a  streptococcus.  The 
streptococcus  most  often  found  differs  in  some  respects  from 
streptococcus  pyogenes.  It  has  been  referred  to  as  a  strepto- 
diplococcus  because  the  chain  arrangement  depends  upon  a 
succession  of  paired  cocci ;  but  this  appearance  is  common  to 
the  majority  of  streptococci.      It  is  feebly  Gram-positive  or 


312  CLINICAL   PATHOLOGY. 

even  Gram -negative,  and  is  of  very  low  virulence  to  animals. 
The  predominance  of  a  streptococcus  in  cases  of  perforated 
ulcer  helps  to  distinguish  them  from  lesions  lower  in  the  gut, 
where  the  exudate  is  almost  entirely  bacillary.  A  very  similar 
streptococcus  is  often  present  in  the  pus  of  a  perinephric 
abscess.  It  must  not  be  supposed  that  the  presence  of  a 
streptococcus  in  the  peritoneum  in  a  case  of  perforated 
gastric  ulcer  indicates  that  the  ulcer  was  due  to  streptococcal 
inflammation.  Streptococci  of  a  similar  nature  may  be  found 
frequently  in  the  gastric  contents  in  the  absence  of  ulceration, 
and,  whatever  may  be  the  cause  of  gastric  ulcer,  the  effect  of 
perforation  is  to  give  to  the  organisms  which  may  have  been 
in  the  stomach  access  to  the  peritoneum.  The  same  warning 
must  be  applied  to  all  bacteriological  finding  in  peritonitis 
following  visceral  lesions.  Among  other  organisms  which  may 
be  found  in  this  condition  are  staphylococci  and  B.  coli. 

Duodenal  ulcer. — The  bacteriology  of  the  peritoneum 
after  perforation  of  a  duodenal  ulcer  is  similar  to  the  fore- 
going, but  streptococci  are  less  commonly,  and  staphylococci 
and  colon  bacilli  more  commonly,  met  with. 

The  ileum, — The  most  important  variety  of  perforation  of 
the  ileum  is  that  which  follows  a  typhoid  ulcer,  the  most 
common  site  of  perforation  being  within  the  last  foot  of  the 
ileum.  Failure  to  find  the  typhoid  bacillus  in  these  cases  is 
common,  and  is  no  evidence  against  the  specific  cause  of 
the  intestinal  ulceration.  Colon  bacilli,  staphylococci,  and 
streptococci  are  recovered  more  frequently  than  the  typhoid 
bacillus,  and  possibly  in  the  majority  of  cases  take  a  greater 
share  in  the  peritoneal  inflammation.  Similar  organisms 
(with  the  exception  of  B.  typhosus)  are  found  in  the 
peritoneum  after  perforation  of  the  ileum  from  injury  or 
other  causes. 

The  appendix. — In  cultures  made  from  the  lumen  of  an 
inflamed  appendix,  from  a  localised  appendix  abscess,  from  the 
general  peritonitis  accompanying  a  diseased  or  perforated 
appendix,  and  from  the  residual  and  remote  abscesses  which 
may  follow  appendicitis  the  predominant  organism  found  is  in 
the  great  majority  of  cases  the  colon  bacillus.  It  is  open  to 
doubt,  however,  whether  anything  approaching  this  propor- 
tion of  appendicular  inflammation  is  produced  by  the  same 
organisms  which  cause  the  peritonitis.     There  is  no  evidence 


PANCEEAS  -LIVER— SPLEEN— PERITONEUM.     313 

of  any  specific  cause  of  appendicitis,  and  it  is  possible  that  a 
variety  of  organisms  may  be  responsible,  and  that  as  soon  as 
a  communication  has  been  established  between  the  lumen 
of  the  gut  and  the  peritoneal  cavity  the  colon  bacillus  rapidly 
multiplies  and  takes  the  predominant  share  in  the  subsequent 
peritonitis.  There  is  abundant  evidence  that  the  colon  bacillus 
in  pure  culture  is  able  to  set  up  a  virulent  peritonitis.  In 
addition  to  the  colon  bacillus  other  organisms  are  frequently 
present  in  film  preparations.  Long,  thin  and  sometimes 
beaded  bacilli  are  often  found,  and  are  probably  the  cause  of 
the  offensive  smell  so  often  met  with  in  the  pus  of  an  appendix 
abscess.  These  organisms  do  not  grow  in  aerobic  cultures,  and 
not  always  under  anaerobic  conditions.  They  are  probably 
identical  with  the  organisms  found  in  stinking  empyemata 
and  in  some  cerebral  abscesses,  and  their  role  appears  to  be 
mainly  that  of  saprophytes.  Their  normal  habitat  is  the 
alimentary  or  respiratory  tract.  Staphylococci  and  strepto- 
cocci are  commonly  met  with  in  film  preparations  in  association 
with  the  colon  bacillus,  particularly  in  localised  appendix 
abscesses.  They  may  also  be  isolated  in  culture,  and  are 
occasionally  present  in  pure  culture. 

The  bacillus  pyocyaneus  is  less  frequently  met  with,  and  is 
practically  never  found  in  association  with  the  colon  bacillus. 
The  latter  organism  appears  to  be  unable  to  exist  in  the 
presence  of  pyocyaneus. 

Lesions  of  the  large  intestine  may  originate  a  peritonitis 
as  after  perforation  of  a  simple  or  a  malignant  ulcer.  The 
organisms  found  in  the  peritoneal  exudate  are  of  much  the 
same  type  as  those  met  with  in  appendix  cases. 

The  pelvic  organs  in  the  female  may  cause  peritonitis 
following  suppuration  in  one  of  them.  A  pyosalpinx  rarely 
ruptures,  but  when  it  does  peritonitis  usually  follows.  The 
pus  is  almost  invariably  sterile,  and  very  occasionally  the 
gonococcus  can  be  identified  in  film  preparations  after  con- 
siderable search.     Recovery  in  such  cases  is  the  rule. 

In  the  case  of  suppuration  in  ovarian  cysts  or  tumours  a 
peritonitis  of  the  ordinary  type  is  set  up,  and  colon  bacilli, 
streptococci,  and  staphylococci  are  found  in  the  exudate.  Not 
infrequently  the  pneumococcus  is  isolated  in  pure  culture 
from  the  pus  in  the  neighbourhood  of  an  ovarian  abscess,  and 
the  prognosis  appears  to  be  favourable. 


314  CLINICAL   PATHOLOGY. 

Post  operative  peritonitis  is  now  fortunately  of  rare 
occurrence,  but  even  in  the  most  careful  hands  occasional 
instances  of  operative  infection  occur.  In  the  pus  of  such 
cases  the  streptococcus  pyogenes  may  be  isolated  in  pure 
culture  and  a  rapidly  fatal  course  is  to  be  expected.  Less 
virulent  staphylococci  are  found  in  some  cases,  and  in  others 
colon  bacilli  or  B.  pyocaneus. 

Intestinal  obstruction. — The  commonest  variety  of 
intestinal  obstruction  is  that  which  follows  strangulation  of 
a  hernia.  In  cultures  made  from  the  general  peritoneal 
cavity  staphylococci  are  frequently  obtained,  and  less  com- 
monly colon  bacilli.  The  more  damaged  the  gut,  the  more 
frequently  are  colon  bacilli  obtained,  and  the  more  serious 
is  the  prognosis.  A  considerable  amount  of  free  fluid  is  often 
found  in  the  hernial  sac.  In  the  majority  of  such  cases  this 
fluid  is  quite  clear,  contains  few  cells  other  than  endothelials, 
and  cultures  from  it  remain  sterile.  If  the  gut  in  the  sac  is 
gangrenous,  organisms  are  more  likely  to  be  obtained  in 
culture. 

In  cases  of  obstruction  due  to  other  causes  the  probability 
of  obtaining  sterile  cultures  from  the  peritoneum  depends 
upon  the  chronicity  of  the  process.  The  more  acute  the  case, 
the  more  commonly  are  organisms  found. 

"Idiopathic  peritonitis." — This  term  was  formerly 
applied  to  cases  of  peritoneal  inflammation  without  any 
recognisable  focus  in  the  intestine.  An  important  organism 
met  with  in  such  cases,  and  often  in  pure  culture,  is  the 
pneumococcus.  Pneumococcal  infection  of  the  peritoneum  is 
more  common  in  children  than  in  adults,  and  females  are 
more  frequently  affected  than  males.  A  pneumococcal  infection 
of  the  lung  may  be  present,  but  more  commonly  no  such  local 
focus  is  found,  and  the  organism  appears  to  have  entered  the 
peritoneum  from  the  general  circulation  in  much  the  same 
way  as  it  may  enter  a  synovial  cavity.  In  young  girls  the 
pneumococci  appear  in  a  certain  percentage  of  cases  to  enter 
by  way  of  the  genital  tract.  The  organisms  may  be  present 
in  pure  culture  in  the  interior  of  the  uterus,  and  are  among 
the  causative  bacteria  to  be  met  with  in  cases  of  ovarian 
suppuration. 

Streptococcal  peritonitis  due  to  causes  other  than  intestinal 
may    develop    as    a    terminal   infection    in    several   chronic 


PANCREAS— LIVER— SPLEEN— PERITONEUM.     315 

diseases,  of  which  chronic  nephritis  is  the  most  common. 
Peritonitis  may  also  develop  as  part  of  a  general  streptococcal 
septicaemia  with  a  primary  focus  elsewhere.  More  rarely  a 
streptococcal  peritonitis  may  be  present  as  a  local  disease  and 
without  any  traceable  source  of  infection. 

The  prognosis  in  those  cases  in  which  pneumococci  are 
found  in  the  peritoneum  is  much  more  favourable  than  those 
associated  with  streptococci.  The  streptococcal  cases  are 
almost  invariably  fatal. 

Tuberculous  peritonitis.  —  In  tuberculous  peritonitis 
with  effusion  the  fluid  is  commonly  quite  clear,  and  the  pre- 
dominant cell  is  a  small  lymphocyte.  Mixed  cellular  fluids 
are,  however,  common  in  the  peritoneal  cavity,  and  a  percentage 
of  lymphocytes  is  found  in  other  than  tuberculous  conditions. 
Secondary  infection  of  a  tuberculous  peritonitis  is  fairly 
frequent,  and  the  fluid  may  be  turbid  or  purulent  and  con- 
tain a  considerable  percentage  of  polynuclear  cells.  Staphy- 
lococci are  often  found  in  cultures  taken  from  such  cases. 

Tubercle  bacilli  are  usually  difficult  to  find,  but  may  be  very 
numerous,  and  should  be  sought  for  by  the  ordinary  methods. 
If  the  bacilli  cannot  be  found  the  fluid  can  be  proved  to  be 
tuberculous  by  injection  into  a  guinea-pig. 


CHAPTEK  XXII. 


THE    F.^CES. 


It  is  remarkable  how  much  the  clinical  investigation  of  the 
faeces  has  fallen  behind  the  physiological  knowledge  of  the 
excreta.  Notwithstanding  the  interest  attached  to  numerous 
intestinal  disorders  and  the  importance  attributed  to  the  con- 
dition known  as  intestinal  toxaemia,  the  usual  investigation  of 
the  stools  consists  of  little  more  than  a  perfunctory  inspection. 
The  main  reason  of  this  neglect  is  that  very  few  simple 
clinical  investigations  are  known  to  throw  any  direct  light  on 
the  condition  of  the  patient.  Elaborate  chemical  analyses  of 
the  excreta  require  that  the  patient  should  be  dieted  in  a  manner 
impossible  in  the  ordinary  hospital  ward,  and  the  analyses 
themselves  are  too  prolonged  for  ordinary  routine  work.  A 
certain  number  of  minor  investigations,  however,  such  as  are 
within  the  scope  of  every-day  work,  are  of  considerable  use  in 
some  diseases  and  essential  for  diagnosis  in  others. 

Such  methods  of  examination  as  are  given  here  are  divided 
into  naked-eye,  chemical,  microscopical,  and  parasitological 
investigations. 

In  any  investigation  it  is  most  advisable  that  the  ordinary 
stool  of  the  patient  should  be  examined  when  available,  and 
not  the  stool  which  follows  the  administration  of  a  purgative 
or  the  giving  of  an  enema. 

The  naked-eye  investigation. — The  amount  and  the 
appearance  of  the  f  aeces  naturally  vary  considerably  in  health. 
The  quantity  passed  by  a  healthy  man  on  an  average  diet  in 
the  24  hours  is  said  to  be  about  150  grammes. 

The  colour  of  the  stools  is  very  variable,  and  the  colour  in 
health  is  due  to  the  presence  of  the  pigment  stercobilin. 
Stercobilin  is  very  similar  to  the  urinary  pigment  urobilin, 
and  is  considered  to  be  identical  with  it.  The  two  bodies  give 
the  same  chemical  tests.  Urobilin,  or  a  very  similar  sub- 
stance, can  be  artificially  produced  from  bile  pigment,  and 
there  is  little  doubt  that  the  stercobilin  of  the  faeces  is  formed 
from  bilirubin  in  the  alimentary  canal.    Unaltered  bile  pigment 


THE    FAECES.  317 

is  never  present  in  the  faeces  in  health.  The  colour  of  the 
faeces  may  be  darkened  by  an  excess  of  stercobilin,  or  after 
the  taking  of  medicines  containing  iron,  manganese,  or  bismuth. 
The  blackening  of  the  stools  as  the  result  of  drugs  is  due  to  the 
formation  of  the  sulphides  of  the  metals  in  the  alimentary 
tract.  Blood  coming  from  the  lower  part  of  the  bowel  may 
be  of  the  usual  red  colour,  but  if  from  the  upper  part  it  is 
black,  and  the  stools  resemble  closely  those  which  follow  the 
taking  of  iron  or  bismuth  and  can  only  be  distinguished  by 
a  chemical  examination.  Green  stools  may  under  abnormal 
circumstances  be  due  to  the  presence  of  unaltered  bile  pig- 
ment. Yellow  stools  follow  the  administration  of  senna, 
santonin,  and  rhubarb.  The  colour  of  the  faeces  is  lost  if  the 
bile  is  prevented  from  entering  the  intestinal  canal,  and  the 
characteristic  "  clayey  "  stools  of  jaundice  are  passed.  Light- 
coloured  stools,  due  to  a  diminution  of  stercobilin,  are  passed  in 
various  conditions  associated  with  diarrhoea.  They  do  not 
necessarily  contain  an  excess  of  fat,  but  cannot  be  distinguished 
by  the  naked  eye  from  the  "  fatty "  stools  suggestive  of 
pancreatic  disease. 

The  odour  of  the  faeces  is  due  mainly  to  the  presence  of 
indole  and  scatole.  The  offensiveness  varies  with  the  nature 
of  the  food — being  much  greater  with  a  meat  than  a  carbo- 
hydrate diet — and  with  the  amount  of  intestinal  putrefaction. 

The  consistence  of  the  stool  naturally  passed  is  of  great 
importance.  The  formed  stool  of  health  is  replaced  by  the 
more  or  less  watery  evacuation  of  diarrhoea  from  a  large 
number  of  causes.  An  unformed  stool  in  an  adult  on  an 
ordinary  diet  is  always  abnormal,  irrespective  of  the  number 
of  the  motions  in  the  24  hours. 

Abnormal  ingredients.  —  Numerous  abnormal  sub- 
stances in  the  stools  can  be  detected  by  the  ordinary  inspec- 
tion. In  cases  where  abnormal  substances  are  to  be  expected 
the  examination  is  greatly  facilitated  by  repeatedly  washing 
the  stool  in  tap  water  through  a  tine  sieve  and  inspecting  the 
residue.  The  following  are  among  the  abnormal  substances 
to  be  looked  for. 

The  larger  animal  parasites  are  easily  detected  with  the 
naked  eye.     They  will  be  described  subsequently. 

Mucous  shreds  and  mucous  casts  are  of  considerable 
importance,  and  are  readily  seen  in  the  residue  of  the  stool 


318  CLINICAL   PATHOLOGY. 

floated  in  water.  They  are  commonly  met  with  in  numerous 
varieties  of  colitis,  and  consist  as  a  rule  of  thin,  ragged  mem- 
branes of  varying  size  composed  of  mucin  and  fibrin.  Large 
mucous  casts  of  the  alimentary  tract  are  less  commonly  seen, 
and  may  appear  as  long,  twisted  strands  which  bear  some 
resemblance  to  tape-worms.  The  strands  are  not  regularly 
segmented,  and  should  be  readily  distinguished  from  parasites. 
Portions  of  undigested  food,  and  particularly  orange  and 
banana  fibre,  closely  resemble  mucoid  shreds,  but  can  be 
differentiated  under  the  microscope.  Small  balls  of  banana 
fibre  have  some  microscopic  resemblance  to  worm  segments 
filled  with  ova. 

Shreds  of  epithelium,  and  rarely  particles  of  malignant 
growth,  may  be  met  with  and  should  be  reserved  for  micro- 
scopical examination. 

Pus  and  blood  may  be  present  in  amount  obvious  to  the 
naked  eye,  but  in  nearly  all  cases  the  macroscopic  evidence 
should  be  confirmed  by  other  means. 

Gall-stones  are  to  be  carefully  looked  for  in  all  suspicious 
cases,  and  are  readily  extracted  from  the  faeces  by  the  sieve  pro- 
cess described  above.  Gall-stones  have  to  be  distinguished 
from  scybalous  particles  of  faeces,  and  after  washing  in  water 
they  must  be  chemically  tested  for  cholesterin.  Cholesterin 
is  normally  present  in  the  faeces,  but  a  solid  macroscopic 
object  in  the  faeces  consisting  almost  entirely  of  this  substance 
is  certainly  derived  from  the  gall  bladder.  Faecal  concretions 
are  as  a  rule  more  friable  and  pultaceous,  contain  very  little 
or  no  cholesterin,  and  often  a  considerable  amount  of  car- 
bonates or  phosphates. 

Intestinal  sand  is  occasionally  passed  in  considerable 
amounts  in  some  forms  of  colitis.  True  intestinal  sand  is  of 
a  brownish  colour,  and  is  composed  mainly  of  calcium  car- 
bonate and  calcium  phosphate.  Cholesterin  is  absent.  The 
colour  is  due  to  stercobilin  with  often  traces  of  bile  pigment. 
False  intestinal  sand  consists  as  a  rule  of  particles  of  undigested 
vegetable  fibres,  and  most  commonly  follows  the  ingestion  of 
pears. 

Chemical  examinations. — The  reaction. — The  reaction 
of  the  stool  to  litmus  paper  has  no  particular  significance. 
The  normal  stool  may  be  either  faintly  acid  or  faintly  alkaline. 
Marked  acidity  or  alkalinity  is  abnormal. 


THE   F.ECES.  319 

Stercobilin  is  probably  identical  with  urobilin,  and  can  be 
detected  in  the  faeces  as  follows  : — 

To  a  watery  extract  of  the  faeces  add  sulphuric  acid  in  the 
proportion  of  2  grammes  to  the  litre,  and  solid  ammonium 
sulphate.  Filter  and  wash  the  precipitate  with  warm,  saturated 
ammonium  sulphate  solution. 

Dry  on  a  water  bath. 

Extract  with  a  boiling  alcoholic  solution  of  ammonia. 

To  a  portion  of  the  extract  add  a  few  drops  of  a  10  per  cent, 
solution  of  zinc  chloride.  The  solution  becomes  fluorescent 
in  the  presence  of  stercobilin. 

Acidify  another  portion  with  acetic  acid,  and  examine 
spectroscopically  for  the  urobilin  spectrum  (page  224). 

A  simpler  and  more  direct  method  is  the  following : — 

Extract  a  portion  of  the  fasces  with  chloroform. 

Pour  off  the  tinted  extract,  and  add  hydrochloric  acid  con- 
taining a  little  nitric  acid. 

Examine  with  the  spectroscope. 

The  chloroform  in  this  process  extracts  the  chromogen,  and 
the  acid  mixture  converts  the  chromogen  into  stercobilin  (or 
urobilin).  Stercobilin  may  by  these  tests  be  demonstrated  even 
hi  almost  colourless  stools. 

Stercobilin  is  absent  in  cases  of  complete  occlusion  of  the 
bile  ducts.  It  is  present  if  the  occlusion  is  incomplete,  how- 
ever deeply  jaundiced  the  patient  may  be.  The  test  is  thus  a 
satisfactory  one  for  the  demonstration  of  complete  occlusion 
of  the  bile  ducts. 

Stercobilin  is  also  stated  to  be  usually  absent  in  carcinoma 
of  the  pancreas. 

Bile. — Bile  pigment  is  normally  absent  from  the  faeces.  It 
is  present  in  some  cases  of  diarrhoea,  and  traces  are  found  in 
conditions  associated  with  jaundice. 

Bile  pigment  may  be  tested  for  by  Gmelin's  test  applied  to  a 
watery  mixture  of  the  faeces,  or  a  particle  of  faeces  may  be  mixed 
with  a  concentrated  solution  of  mercuric  chloride,  covered  and 
allowed  to  stand  for  24  hours.  The  normal  stool  coloured  by 
stercobilin  turns  red.  If  bile  pigment  is  present  green  particles 
appear. 

Bile  acids  may  be  tested  for  as  follows  : — 
Extract   the  faeces  with  alcohol.     Dissolve  the  residue  in 
dilute  caustic  soda  and  perform  Pettenkofer's  test,  thus  : — 


320  CLINICAL   PATHOLOGY. 

Dissolve  a  fragment  of  cane  sugar  in  the  solution  of  the 
residue  in  a  test  tube. 

Kun  in  about  5  c.c.  of  concentrated  sulphuric  acid  to  the 
bottom  of  the  tube  and  gently  shake. 

A  slowly-developing  purple  colour  forms  from  the  line  of 
junction  of  the  two  fluids. 

Excess  of  cane  sugar  in  the  mixture  must  be  avoided,  since 
it  is  charred  by  the  acid  and  so  masks  the  reaction. 

Blood  pigment. — Blood  present  in  considerable  amount 
and  coming  from  the  lower  part  of  the  tract  may  be  detected 
by  the  microscope  or  by  its  spectrum. 

Blood  in  small  amount  or  from  the  upper  part  of  the  tract 
cannot  be  recognised  under  the  microscope,  and  is  best 
examined  for  by  one  of  the  tests  for  so-called  "  occult  "blood. 
By  occult  blood  is  meant  blood  present  in  minute  traces,  such 
as  may  come  from  an  oozing  gastric,  duodenal,  or  malignant 
ulcer.  Various  delicate  tests  capable  of  revealing  very  minute 
traces  of  blood  are  employed,  and  are  of  some  assistance  in 
the  diagnosis  of  alimentary  conditions  associated  with  haemor- 
rhage. Before  performing  such  a  test  it  is  necessary  to  put 
the  patient  on  a  blood- free  diet  for  48  hours. 

The  test  may  be  performed  as  follows : — 

Rub  up  a  fragment  of  faeces  in  4  c.c.  of  water  in  a  mortar. 

Or,  if  the  faeces  are  liquid,  mix  thoroughly  2  c.c.  of  faeces  and 
2  c.c.  of  water. 

Add  2  c.c.  of  glacial  acetic  acid. 

Pour  into  a  test  tube  and  shake  thoroughly. 

Add  5  c.c.  of  ether. 

Invert  the  tube  slowly  and  gently  twenty  times ;  if  the  inver- 
sion is  roughly  done  the  ether  will  not  separate  on  standing. 

Allow  to  stand  and  pour  off  2  c.c.  of  the  ether  extract  into 
a  10  c.c.  measure. 

Test  for  haematin  acetate  thus  : — 

Add  \  c.c.  of  glacial  acetic  acid. 

Add  2  c.c.  of  a  saturated  solution  of  benzidin  in  rectified 
spirit.     The  benzidin  solution  must  be  freshly  prepared. 

Add  2  c.c.  of  a  five- volume  solution  of  hydrogen  peroxide. 

Mix. 

In  the  presence  of  blood  the  mixture  becomes  blue  of  a  greater 
or  less  intensity.  With  a  very  minute  trace  of  blood  the 
colour  is  greenish. 


THE   F^CES.  321 

Albumin. — In  health  no  proteid  reaction  can  be  obtained 
in  the  faeces  on  an  ordinary  diet,  but  a  considerable  quantity 
of  albumin  can  be  detected  in  the  stools  in  numerous  conditions 
associated  with  diarrhoea. 

To  test  for  albumin  : — 

Add  to  a  small  portion  of  fasces  a  considerable  quantity  of 
water  acidified  with  acetic  acid. 

Mix  thoroughly,  and  allow  to  stand. 

Filter  several  times. 

Test  the  filtrate  for  albumin  by  the  ordinary  tests. 

Fat. — The  normal  faeces  contain  both  neutral  fats  and  fatty 
acids.  The  determination  of  the  amount  of  fat  in  the  stools 
and  the  form  in  which  it  is  present  may  be  of  assistance  in 
numerous  forms  of  intestinal  disorder,  and  is  most  frequently 
required  in  the  investigation  of  pancreatic  disease.  It  will  be 
remembered  that  the  action  of  the  pancreatic  juice  upon  the 
neutral  fats  in  the  duodenum  is  to  split  them  into  fatty  acids 
and  glycerin,  a  process  of  hydrolysis  known  as  saponification. 
The  saponification  is  aided  by  the  admixture  of  bile.  The 
flow  of  the  pancreatic  juice  is  determined  by  secretin,  which 
is  produced  in  the  duodenal  mucous  membrane  on  contact 
of  the  acid  contents  of  the  stomach.  The  secretin  passes 
into  the  blood  and  is  carried  to  the  pancreas.  The  fat-splitting 
ferment  of  the  pancreas  is  of  course  the  lipase. 

The  fatty  acids  split  off  during  saponification  combine  in 
the  intestine  with  the  alkalies  to  form  soaps.  The  soaps  are 
the  sodium  and  potassium  salts  of  the  higher  fatty  acids,  and 
become  reconverted  into  fatty  acids  on  the  addition  of  an 
inorganic  acid. 

The  fatty  acids  or  soaps  are  absorbed  from  the  intestine,  and 
are  built  up  again  into  fats  in  the  tissues.  The  neutral  fats 
and  free  fatty  acids  are  soluble  in  ether;  the  soaps  are 
insoluble  in  ether. 

These  elementary  chemical  points  are  necessary  for  the 
understanding  of  the  methods  of  estimating  fatty  bodies  in  the 
fasces  and  the  interpretation  of  the  results. 

The  simple  method  of  estimating  the  fats  described  by 
Cammidge  is  sufficiently  accurate  for  clinical  purposes,  and  is 
advocated  by  him  as  a  means  of  diagnosis  in  biliary  and  pan- 
creatic diseases. 

Under  normal  conditions  the  total  fat  forms  from  20  to  25 

p.  21 


322  CLINICAL   PATHOLOGY. 

per  cent,  of  the  dried  faeces,  and  consists  of  neutral  fat,  and 
soaps  representing  the  free  fatty  acids,  in  about  equal  pro- 
portions. 

If  the  entry  of  the  pancreatic  secretion  into  the  duodenum  is 
prevented,  as  in  pancreatic  calculus  or  carcinoma  of  the  pan- 
creas, or  if  the  pancreatic  secretion  is  poor,  as  in  chronic 
pancreatitis,  the  fats  are  imperfectly  dealt  with ;  consequently 
an  excess  of  fat  is  found  in  the  stools,  and  since  the  neutral 
fats  have  been  insufficiently  split  up  by  the  deficient  pancreatic 
juice,  they  are  present  in  excess  of  the  fatty  acids. 

If  the  biliary  secretion  is  interfered  with  and  the  pancreatic 
secretion  is  normal,  the  neutral  fats  become  converted  into 
fatty  acid  and  glycerin,  as  under  normal  conditions :  but 
saponification  and  absorption  of  fat  is  interfered  with ;  conse- 
quently the  total  fat  is  again  in  excess,  but  the  fatty  acids  are 
in  excess  of  the  neutral  fats.  A  similar  state  prevails  in  some 
varieties  of  diarrhoea  and  in  other  conditions  which  interfere 
with  intestinal  absorption. 

The  estimation  of  the  fats  is  performed  as  follows : — 

A  sample  of  the  usual  stool  is  taken,  the  patient  being  given 
an  ordinary  mixed  diet. 

The  faeces  are  placed  in  a  porcelain  evaporating  dish  and 
heated,  first  over  the  water  bath,  and  finally  on  the  sand  bath 
in  a  fume  cupboard.  The  faeces  are  stirred  occasionally  with 
a  glass  rod  and  the  heating  process  is  continued  until  they 
are  thoroughly  dry.  The  process  takes  some  hours  as  a  rule, 
and  may  be  completed  by  leaving  in  a  drying  chamber  over 
night.     When  cool  the  dried  faeces  are  practically  inoffensive. 

The  dried  faeces  are  powdered  into  as  fine  a  dust  as  possible 
in  a  mortar.  It  may  be  impossible  to  powder  very  fatty 
stools. 

Two  samples,  each  of  0*5  gramme,  are  carefully  weighed 
out. 

Two  clean  and  absolutely  dry  Schmidt- Werner  tubes  are 
prepared,  and  labelled  A  and  B.  Each  should  be  provided 
with  a  10  c.c.  mark. 

Into  the  lower  bulb  of  each  place  0*5  gramme  of  faeces. 

Wash  the  residue  into  the  A  tube  with  1  in  3  hydrochloric 
acid,  and  fill  up  with  the  acid  to  the  10  c.c.  mark. 

Wash  the  residue  into  the  B  tube  with  distilled  water,  and  fill 
with  water  to  the  10  c.c.  mark. 


THE   F^CES.  323 

In  each  tube  all  the  faeces  must  be  collected  with  the  fluid 
in  the  lower  bulb. 

The  A  tube  is  then  heated  in  boiling  water  for  15  minutes 
and  is  rotated  from  time  to  time.  By  this  process  the  fatty- 
acids  are  split  off  from  their  bases  and  rendered  soluble  in 
ether. 

Cool  the  A  tube. 

Fill  both  tubes  to  the  50  c.c.  mark  with  ether,  and  cork  them 
securely. 

Slowly  invert  and  rotate  each  tube  forty  times. 

Allow  the  tubes  to  stand  for  30  minutes  or  until  all  the 
residue  has  collected  into  the  lower  bulbs. 

While  the  tubes  are  standing  weigh  carefully  two  small  dry 
evaporating  flasks  labelled  A  and  B.  Note  the  weight  of  each. 
Into  each  appropriate  flask  measure  20  c.c.  of  the  clear  ethereal 
extract  from  the  tubes  :  the  measurement  is  most  conveniently 
made  with  a  pipette,  but  care  must  be  taken  to  keep  the  nozzle 
of  the  pipette  under  the  surface  of  the  fluid. 

Note  the  amount  of  ether  left  in  each  tube. 

Evaporate  the  ether  extracts  by  holding  the  flasks  in  a 
stream  of  hot  water. 

Dry  the  residue  by  heating,  without  charring,  on  a  water 
bath  in  the  fume  cupboard.  Unless  this  step  is  thoroughly 
done  the  error  in  the  subsequent  estimation  is  considerable. 

After  cooling  weigh  each  flask. 

The  difference  in  -the  weight  of  each  represents  the  weight 
of  fat  extracted  by  20  c.c.  of  ether  from  0'25  gramme  of  dried 
faeces,  supposing  no  ether  to  have  been  lost  from  the  tubes 
and  consequently  the  level  of  the  remaining  fluid  to  be  at  the 
30  c.c.  mark. 

The  percentage  of  fat  in  the  dried  faeces  is  thus  given  by 
multiplying  the  result  by  400. 

The  yield  from  the  A  tube  may  be  reckoned  as  the  total  fat, 
since  it  includes  the  ether-soluble  neutral  fats  and  the  fatty 
acids  which  have  been  split  off  from  their  bases  by  the  hydro- 
chloric acid. 

The  yield  for  the  B  tube  may  be  reckoned  as  neutral  fat. 

The  difference  between  the  two  tubes  represents  the  fatty 
acids. 

In  13  cases  of  carcinoma  of  the  pancreas  in  which  the  faeces 
were  thus  analysed  by  Cammidge  the  total  fat  ranged  from 

21—2 


324  CLINICAL   PATHOLOGY. 

50  to  90  per  cent,  of  the  dried  fasces,  and  of  this  the  neutral  fat 
formed  40  to  60  per  cent.,  and  the  fatty  acids  9  to  33  per  cent. 

An  estimate  of  the  amount  of  fat  present  in  the  fasces  from 
a  naked-eye  inspection  is  extremely  misleading,  and  often 
the  so-called  fatty-looking  stools  of  alleged  pancreatic  diarrhoea 
are  found  to  be  merely  pallid  stools  with  a  deficiency  of 
stercobilin  and  a  normal  amount  of  fat. 

Some  chemical  estimation  is  required,  and  the  method  of 
Cammidge  requires  little  apparatus,  takes  up  much  less  actual 
time  than  might  be  supposed  from  the  above  description,  and  is . 
sufficiently  accurate  to  detect  the  gross  changes  which  may 
occur  in  disease.  The  deductions  to  be  drawn  from  the 
results  are  to  be  carefully  correlated  with  the  clinical  evidence 
and  all  other  available  modes  of  examination.  Taken  by  them- 
selves estimations  of  this  character  are  of  very  little  value. 

Other  chemical  investigations. — Mucin  appears  to  be 
a  normal  constituent  of  the  stools.  It  may  be  tested  for  by 
adding  to  a  watery  suspension  of  the  stool  an  equal  volume  of 
lime-water.  The  mixture  is  allowed  to  stand  until  the  next 
morning  and  is  then  filtered.  A  little  acetic  acid  is  added 
to  the  filtrate  and  a  white  cloud  of  mucin,  insoluble  in  excess 
of  acid  and  increased  on  boiling,  is  produced. 

Peptone  is  absent  from  the  fasces  in  health,  but  is  present 
usually  in  cases  of  typhoid  fever,  and  in  all  stools  containing 
pus.  Peptone  is  absent  in  catarrhal  jaundice,  but  usually 
present  in  cirrhosis  and  malignant  disease  of  the  liver. 

Peptone  in  the  faeces  may  be  detected  as  follows :  — 

Mix  some  of  the  fasues  with  water. 

Boil  and  filter  while  hot. 

Test  for  albumin — if  absent, 

Mix  the  filtrate  with  neutral  lead  acetate  and  filter  after 
standing. 

Acidulate  filtrate  with  hydrochloric  acid. 

Add  phospho-tungstic  acid  until  no  more  precipitate  forms. 

Filter  at  once. 

Wash  the  precipitate  on  the  filter  with  5  per  cent,  sulphuric 
acid  until  a  colourless  filtrate  is  obtained. 

Wash  the  precipitate  off  the  filter  paper  into  a  dish  with  the 
minimum  amount  of  water. 

Mix  the  filtrate  with  barium  carbonate  till  alkaline. 

Heat  on  a  water  bath  for  15  minutes. 


THE   MCES.  325 

Test  for  peptone  by  the  biuret  test  as  follows  : — 

Add  caustic  potash  and  drop  by  drop  a  10  per  cent,  solution 
of  copper  sulphate. 

A  bluish  pink  or  violet  colour  indicates  peptone. 

If  albumin  is  present  in  the  original  nitrate  it  must  be 
removed  as  follows  : — 

A  solution  of  acetate  of  soda  is  added,  and  next  one  of 
perchloride  of  iron.  The  mixture  is  then  exactly  neutralised 
with  caustic  soda,  boiled,  filtered,  and  allowed  to  cool. 

Hydrochloric  acid  is  then  added  and  the  process  described 
above  is  performed. 

Cholesterin  is  present  in  the  faeces  in  health  and  can 
be  extracted  from  them.  It  practically  never  occurs  in  a 
crystalline  form. 

Microscopical  investigation.— Numerous  points  of  con- 
siderable importance  may  be  found  in  the  course  of  a  micro- 
scopic examination  of  the  faeces. 

In  order  to  make  the  examination  prepare  in  a  test  tube  a 
turbid  suspension  of  the  faeces  in  normal  saline.  Shake 
thoroughly.  Allow  the  suspension  to  stand.  Pipette  up  the 
deposit,  and  examine  it  on  a  slide  with  a  cover-slip  over  it  in 
exactly  the  manner  employed  for  a  urinary  deposit. 

If  the  ova  of  intestinal  parasites  are  being  looked  for  a 
similar  mixture  should  be  made  in  a  large  test  tube,  and  a 
little  carbolic  acid  may  be  added  to  remove  the  odour.  After 
standing  the  supernatant  fluid  is  decanted,  and  the  sediment  is 
again  shaken  with  saline.  The  process  is  repeated  two  or 
three  times  and  the  ova  sought  for  in  samples  of  the 
sediment. 

The  general  microscopic  view  of  a  faecal  suspension  is  at  first 
confusing.  The  numerous  particles  of  many  varieties  of  food 
remnants,  and  the  general  debris  lying  among  the  fauna  and 
flora  of  the  intestinal  canal,  provide  somewhat  of  a  diagnostic 
debauch. 

The  following  are  among  the  substances  which  should  be 
looked  for : — 

Vegetable  cells  and  fibres. — These  may  occur  in  a 
great  variety  of  shape  and  size.  Spiral  forms  are  extremely 
numerous,  and  may  be  found  as  free  spirals  or  as  long  cells 
containing  an  evenly-wound  refractile  spiral.  Other  forms 
appear  as  long  and  sejDtate  divisions  marking  off  the  usually 


326  CLINICAL   PATHOLOGY. 

empty  cells.     Some  of  the  cells  may  contain  chlorophyll,  and 
others  starch  granules. 

Starch  granules  should  not  be  present  in  great  numbers ; 
they  may  be  detected  by  their  appearance  and  by  their  blue 
reaction  on  running  a  solution  of  iodine  and  potassium  iodide 
under  the  cover-slip. 

Fat. — Fat  globules  should  only  be  present  in  small  numbers 
in  a  normal  stool.  They  are  present  in  large  quantities  after 
the  injection  of  an  oil  enema.  They  are  readily  recognised  by 
their  shape  and  refractility,  and  if  necessary  by  their  chemical 
reactions.  Fat  occurs  more  commonly  in  the  form  of  sheaves 
of  colourless  pointed  crystals.  These  sheaves  of  fatty  acid 
crystals  dissolve  on  warming,  and  in  ether.  The  soaps  may 
appear  in  the  form  of  coarser  crystals  and  dissolve  on  warming, 
but  not  on  the  addition  of  ether.  When  an  excess  of  fat  is 
present  in  the  faeces  fatty  acid  crystals  are  usually  found,  but 
a  chemical  estimation  is  necessary  in  order  to  determine 
whether  fat  is  actually  in  excess  or  not. 

Muscle  fibres  are  nearly  always  found  in  the  faeces  of  a 
person  on  a  normal  diet.  Their  amount  and  the  degree  of 
their  digestion  naturally  vary  with  the  amount  of  meat 
taken ;  but  under  ordinary  conditions  the  relative  number  of 
undigested  muscle  fibres  is  a  considerable  guide  to  the  activity 
of  the  digestive  juice  and  the  suitability  of  the  diet  to  the 
individual.  Muscle  fibres  as  seen  under  the  microscope  are 
of  a  yellow  colour,  and  are  certainly  recognised  by  their 
fine  transverse  striation.  The  striation  can  be  perfectly  well 
seen  with  a  Jth-inch  objective,  particularly  if  the  diaphragm 
of  the  condenser  is  partly  shut  down.  Under  normal  condi- 
tions few  fibres  will  be  seen  on  any  one  slide,  and  in  the  great 
majority  of  these  the  striae  will  be  almost  or  quite  invisible. 
In  conditions  associated  with  alimentary  disorders  the  fibres 
are  very  numerous  and  their  striation  well  marked. 

Elastic  fibres  can  be  often  detected  in  the  faeces,  and  are 
recognised  by  their  shape,  their  curved  form,  and  their  double 
contour. 

Red  blood  corpuscles  can  as  a  rule  only  be  recognised  in 
the  faeces  under  the  microscope  when  the  haemorrhage  has  come 
from  the  lower  part  of  the  intestinal  canal.  With  quite  pro- 
fuse haemorrhage  from  high  up  in  the  gut,  as  in  duodenal  ulcer 
or  even  in  typhoid  fever,  recognisable  red  cells  are  rarely  met 


THE   F^CES.  327 

with.  Keddish-brown  pigment  masses  are  seen  in  such  cases 
and  can  be  recognised  as  of  probable  blood  origin,  but  the 
chemical  test  for  blood  should  always  be  applied.  The 
sulphides  of  the  metals,  and  particularly  of  bismuth  and  iron, 
give  an  appearanc3  to  the  stools  similar  to  that  produced  by 
altered  blood.  Undsr  the  microscope  the  sulphides  appear  as 
black  amorphous  masses.  Bismuth  may  occasionally  appear 
in  the  stools  in  the  form  of  black  crystals  of  a  shape  similar 
to  that  of  haemin  crystals. 

Leucocytes  are  absent,  or  almost  completely  absent,  from 
the  stools  in  health.  In  the  numerous  conditions  associated 
with  a  catarrhal  state  of  the  intestines  there  is  practically  no 
increase  in  the  number  of  leucocytes  found.  Actual  pus  in 
the  stools  is  very  rarely  seen,  and  any  considerable  increase  in 
the  leucocytes  points  to  actual  ulceration  of  the  gut.  Pus  if 
present  indicates  that  an  abscess  has  discharged  into  the 
intestine,  as  for  example  an  appendix  abscess  into  the  rectum. 
Pus  cells  have  here,  as  elsewhere,  to  be  differentiated  from 
epithelial  cells,  and  in  cases  of  doubt  stained  preparations 
should  be  made. 

Epithelium. — Epithelial  cells,  more  or  less  degenerated, 
are  practically  always  found  in  the  faeces.  They  may  be 
squamous,  or  less  often  columnar,  and  most  commonly  are 
fairly  small,  more  or  less  fusiform  cells.  They  occur  singly  and 
less  often  in  small  plaques.  In  cases  of  intestinal  catarrh 
epithelial  cells  are  usually  much  increased  in  number. 

Crystals. — The  commonest  inorganic  crystal  to  be  found 
is  that  of  calcium  oxalate,  and  the  envelope  forms  are  those 
most  often  seen.  The  crystals  are  derived  from  the  food, 
and  are  most  abundant  after  a  vegetable  diet.  Other  crystals 
which  may  be  present  are  calcium  carbonate  and  calcium 
sulphate  rarely,  calcium  phosphate  and  triple  phosphates 
more  commonly.  These  crystals  are  recognised  in  the  same 
way  as  in  urinary  deposits. 

Bacteria  are  always  present  in  considerable  numbers  in  the 
stools.  The  majority  of  the  organisms  are  bacilli,  and  in 
Gram-stained  films  the  Gram-negative  bacilli  predominate 
in  normal  conditions ;  cocci  are  also  present  as  a  rule.  Spirilla 
and  vibrios  are  less  common,  but  may  appear  in  small  numbers 
in  health.  The  varieties  of  bacteria  and  their  significance  will 
be  considered  in  the  next  chapter. 


328  CLINICAL   PATHOLOGY. 

Ova  of  parasites  are  among  the  abnormal  objects  to  be 
looked  for  in  the  stools.  They  will  be  described  in  the 
account  of  the  intestinal  parasites. 

Debris.— Amorphous  particles  derived  from  the  food  form 
a  considerable  part  of  the  slide  preparations  made  from  the 
faeces.  They  are  of  variable  size,  and  may  appear  as  discrete 
particles  or  in  clumps. 


CHAPTER  XXIII. 

THE    PARASITOLOGY    OF    THE    FiECES. 

The  animal  parasites. — A  considerable  variety  of  animal 
parasites  may  on  certain  occasions  and  in  certain  countries 
infest  the  human  alimentary  tract.  Only  three  species  are, 
however,  commonly  distributed  in  this  country  :  namely, 
Oxyuris  vermicular  is,  Ascai'is  lumbricoides  and  Tcenia  saginata. 
Other  species,  such  as  ankylostoma,  are  common  in  certain 
circumscribed  areas.  Others  again  may  be  imported  by 
their  human  host  from  other  countries,  or  may  occur  only 
fortuitously  in  man. 

The  following  is  a  brief  account  of  the  more  important 
parasites,  arranged  zoologically. 

Platy helminths.  —  These  are  worm-like,  flattened  dorso- 
ventrally,  and  frequently  hermaphroditic. 

They  are  divided  into  three  classes  : — 

Class  1,  Turbellaria,  are  not  parasitic. 

Class  2,  Trematoda  (or  flukes),  are  parasitic  in  man 
and  unsegmented  ;  in  all  the  life-cycle  from  ovum  to  adult  is 
complex,  requiring  an  intermediate  host,  and  asexual  multi- 
plication takes  place  outside  the  body  from  the  sexually- 
produced  egg. 

Class  3,  Cestoda  (tape-worms)  are  parasitic,  are  elongated, 
flattened,  and  segmented  ;  the  mode  of  development  is  com- 
paratively simple. 

Trematodes. — A  considerable  number  of  flukes  are  parasitic 
to  man,  but  none  which  produce  symptoms  are  native  to  this 
country.  The  flukes  may  conveniently  be  divided  on  clinical 
grounds  into  those  which  inhabit  the  biliary  passages,  the 
bronchi,  and  the  blood-vessels. 

Liver  flukes  are  numerous,  the  most  important  being 
Fasciolopsis  buski,  common  in  China  and  India,  and  Clonorchis 
sinensis,  an  extremely  frequent  parasite  of  Eastern  peoples. 
Usually  no  symptoms  are  produced,  but  there  may  be 
enlargement  of  the  liver  with  jaundice  and  ascites.     The  ova 


330 


CLINICAL    PATHOLOGY. 


are  found  in  the  faeces.  They  are  roughly  oval,  but  with  one 
end  narrower  than  the  other,  and  are  of  a  yellowish  colour. 
They  have  a  shell  with  a  lid  at  one  end,  and  contain  round 
retractile  globules. 

The  bronchial  fluke,  or  Paragonimus  westermani,  is 
the  cause  of  endemic  haemoptysis  in  China,  Japan,  and  the 
Philippines.  It  measures  about  10  mm.  long  and  5  mm. 
broad.  Infection  is  diagnosed  by  the  ready  detection  of  the 
ova  in  the  sputuni.  More  rarely  the  parasite  is  found  in  the 
intestine  and  the  ova  in  the  faeces.  The  intermediate  host 
is  probably  a  mollusc,  but  is  undetermined.  The  young 
flukes  or  cercariae  are  probably  taken  into  the  mouth  with 
the  food  and  by  some  unknown  route  reach  the  lungs. 

The  blood  flukes,  of  which  the  most  important  is 
Bilharzia  hcematobia,  are  bisexual  trematodes.    Bilharzia  is  a 

common  parasite  of 
Egypt  and  parts  of 
South  Africa.  The 
adult  worm  lives  in 
the  veins  without  pro- 
ducing much  change  in 
them.  The  symptoms 
are  consequent  upon 
the  passage  of  the  ova 
through  the  mucous 
membrane  into  the 
rectum  or  bladder.  The 
diagnosis  is  determined  by  the  presence  of  the  spined  eggs  in 
the  urine  or  the  faeces.  The  ova  differ  from  those  of  other 
trematodes  in  having  no  lid.  The  single  spine  is  terminal  in 
the  case  of  bladder  infection  and  lateral  in  ova  found  in  the 
faeces.  The  different  ova  may  be  derived  from  two  different 
species  of  parasites.  Ova  may  be  found  some  years  after 
the  patient  has  left  the  infected  district.  They  contain  a 
ciliated  embryo.  The  adult  male  is  from  15  to  18  mm.  long 
and  3  to  5  mm.  broad.  The  female  is  longer  and  thinner, 
being  '20  mm.  long  and  only  0*25  mm.  broad.  The  male 
has  two  suckers,  the  anterior  of  which  is  terminal.  It  is  a 
flat  worm  rolled  longitudinally  upon  itself  to  form  a  hollow 
tube,  within  which  the  female  is  clasped.  Both  life  history  and 
mode  of  infection  are  unknown,  but  there  is  no  question  that 


Fig.  19. — Bilharzia  Hsematobia. 

Ovum.     Male  and  Female.     Natural  Size. 


THE    PARASITOLOGY   OF    THE   FAECES.       331 

water  plays  an  important  part  in  each  case.  The  ciliated 
embryo  in  the  urine  on  reaching  water  bursts  its  capsule  and 
swims  about  busily.  An  intermediate  host  is  probably  neces- 
sary. The  infection  is  thought  to  take  place  by  entry  of  the 
parasite  into  the  urethra  or  anus,  or  possibly  through  the 
skin  while  bathing. 

A  smaller  trematode  also  inhabiting  the  portal  veins  of 
man,  and  common  in  the  East,  is  Schistosomum  japonicum. 
The  male  similarly  contains  the  female  in  a  gynsecophoric 
canal.  The  ova  are  found  in  the  faeces  and  contain  a  ciliated 
embryo,  but  have  no  spine. 

Cestodes. — The  tape-worms  are  divided  into  two  groups  by 
the  character  of  their  heads,  namely,  Dibothriocephaloidea 
and  TceniidcE.  The  heads  of  the  former  are  provided  with 
two  elongated  slits,  the  latter  have  four  round  suckers  and 
a  rostellum,  which  in  some  species  is  armed  with  hooklets. 
All  the  worms  consist  of  a  head  or  scolex,  from  which  arises 
a  series  of  segments  or  proglottides.  Each  proglottis  is 
bisexual. 

The  dibothriocephaloidea. — The  most  important  human 
parasite  of  this  class  is  Bothriocephalus  latus. 

Bothriocephalus  latus  is  not  met  with  in  Great  Britain, 
but  is  common  in  Iceland,  Switzerland,  and  parts  of  Germany. 
The  adult  worm  lives  in  the  intestinal  tract  of  man,  and 
feeds  upon  the  intestinal  contents.  A  severe  anaemia  is  pro- 
duced in  the  host,  probably  by  the  action  of  a  toxin  excreted 
by  the  worm.  The  parasite  is  very  large  and  long,  and  may 
grow  to  25  or  30  feet  in  length.  The  head  is  long  and  narrow, 
and  is  attached  to  the  gut  wall  by  two  long  slit-like  suckers. 
The  genital  pore  opens  upon  the  flat  surface  of  the  pro- 
glottis. The  ova  are  enclosed  in  shells  fitted  with  a  lid,  and 
at  the  time  of  passage  in  the  faeces  are  immature.  The  shells 
are  almost  colourless.  Within  the  ovum  a  six-hooked  embryo 
forms,  with  a  ciliated  capsule  around  it.  Embryo  and  capsule 
are  called  the  oncosphere.  The  ciliated  embryo  escapes  in 
water  and  enters  the  intestinal  tract  of  a  pike  or  other  fish. 
Here  the  ciliated  envelope  disappears,  and  the  embryo  makes 
its  way  through  the  intestinal  wall  into  the  muscles  and, 
losing  its  hooklets,  develops  slit-like  suckers  and  an 
unsegmented  worm-like  body.  This  larval  form  is  eaten  by 
man  or  other  host  and  develops  into  the  adult  segmented  worm. 


332  CLINICAL   PATHOLOGY. 

A  similar  and  even  larger  tape-worm  is  met  with  in  Japan. 
It  may  attain  a  length  of  12  yards,  and  is  known  as 
Diplogonoponcs  grandis. 

The  tseniidse  are  represented  by  several  species  which  may 
be  parasitic  to  man,  and  three  are  common.  Man  is  the 
definitive  host  in  the  case  of  Tcenia  solium  and  Tcenia  saginata, 
but  for  the  former  may  act  as  the  intermediate  host.  Man 
is  the  intermediate  host  for  the  echinococcus.  The  ova  of 
the  tseniidas  ripen  in  the  proglottides,  and  at  the  time  of 
passage  in  the  faeces  are  in  the  oncosphere  stage.  At  this 
stage  the  embryo  has  a  head  armed  with  six  hooklets,  and  is 
contained  in  a  thick,  radially  striated,  but  not  ciliated  capsule. 

The  oncospheres  are  swallowed  by 
man  or  sheep  in  the  case  of  the 
T.  echinococcus,  by  cattle  with  the 
T.    saginata,    and    by   pigs    with 
T.   solium.      The   capsule  is  dis- 
solved in  the  intestinal  canal  of 
the    intermediate    host,    and   the 
hooked   embryo   pierces    the    gut 
wall  and  reaches  the  viscera.    Here 
the  embryo  becomes  encysted,  and 
its  hooklets  drop  off.     The  cyst  or 
Fie.  20.-Head  of  TMa     c3^ercus     becomes    lined    with 
Solium.    Hooklet.    Head      germ    cells>    from    whlch    scollces 
Natural  Size.  develop.     The  echinococcus  cysts 

develop  secondary  or  daughter  cysts, 
and  the  scolices  grow  in  these.  The  cysticercus  is  ingested 
by  a  flesh-eating  animal ;  the  scolices  are  set  free,  attach 
themselves  to  the  gut  wall,  and  grow  into  adult  worms. 

Taenia  solium,  the  pork  tape-worm,  reaches  a  length  of 
about  10  feet.  The  ripe  proglottis  is  about  10  mm. 
long  and  5  mm.  broad.  The  genital  pores,  as  is  the  case 
in  all  the  tseniidse,  open  laterally.  The  uterus  has  about 
10  lateral  branches.  The  rostellum  has  4  suckers  and  a 
double  circle  of  hooklets.  Man  becomes  infected  by  eating 
imperfectly-cooked  and  "  measled "  pork.  The  infection  is 
much  commoner  in  Germany  than  in  Great  Britain.  Man 
occasionally  acts  as  the  intermediate  host,  and  becomes  infected 
by  swallowing  the  oncospheres  ;  such  infection  may  occur  when 
human  faeces  are  used  as  manure,  or  by  auto-infection. 


THE   PARASITOLOGY   OF    THE    F.ECES. 


333 


! 


The  adult  form  can  be  recognised  by  the  passage  of  the  onco- 
spheres and  the  segments  in  the  faeces.  The  head  of  the  worm, 
which  is  a  comparatively  minute  object,  must  be  particularly 
sought  for,  and  the  rostellum,  with  its  suckers  and  hooklets, 
is  readily  recognised  with  a  low  magnification.  The  cyst  con- 
tents of  the  cysticercus  infection  are  searched  for  the  small 
hooklets,  which  are  pointed,  slightly  curved,  and  not  barbed. 
This  cysticercus  is  known  as  the  Cysticercus  celluloses. 

Taenia    saginata   is   the  commonest   of   all  human  tape 
worms.      It  is  larger  and  longer  than  T.   solium    and   may 
attain  a  length  of  20  feet.     The  ripe  proglottis  is  longer  and 
broader  than  that  of    T.  solium.      The   uterus  has  from  20 
to  25  main  branches.     The  head  has  4  suckers  but  no  hook- 
lets, and  there  is  usually  a  more 
or  less  circular  deposit  of  black 
pigment  in  the  anterior  part  of 
the  head.    The  cysticercus  occurs 
in  the  muscles  of  the  ox  and  is 
known  as  Cysticercus  bovis.    Man 
is  probably  never  affected  by  the 
cysticercus,    but    is    always   the 
definitive   host.      Persons    most 
liable  to  be  affected  are  those  who 
eat   raw   or   lightly-cooked  beef, 
the  small  cysticerci  in  which  can 
easily  escape  notice.     The  adult 
worm  in  the  intestine  produces 
few    symptoms   other   than    of    a    subjective    nature.      The 
diagnosis  rests  with  the  detection  of  the  head,  the  proglottides, 
or  the  oncospheres  in  the  fasces. 

Taenia  echinococcus. — The  echinococcus,  or  hydatid,  is 
a  small  tape-worm  only  4  or  5  mm.  long.  It  is  composed 
of  a  variable  number  of  segments,  usually  4,  sometimes  3  or  5. 
The  mature  ova  are  contained  in  the  last  segment.  The  head 
is  provided  with  4  suckers  and  a  double  row  of  hooklets. 
The  adult  tape-worm  is  found  in  dogs,  wolves,  and  jackals ; 
and  the  period  between  ingestion  and  the  passage  of  mature 
ova  is  about  40  days.  The  oncospheres  may  be  deposited  on 
vegetables  or  grass,  and  from  this  source  man  or  sheep  may 
become  infected.  In  man  the  cysticercoid  form  only  is  found, 
and  the  tumours  may  take  three  or  more  years  to  develop. 


Pig.  21.- 

nata. 


Head  of  Taenia  Sagi- 
Head  ^Natural  Size. 


334 


CLINICAL   PATHOLOGY. 


Human  infection  is  common  in  Australia  and  other  sheep- 
raising  countries.  In  England  it  is  perhaps  most  common  in 
the  eastern  counties,  and  a  considerable  number  of  cases  are 
met  with  in  the  Cambridgeshire  district.  Persons  who  have 
never  been  out  of  London  may  occasionally  be  infected, 
possibly  through  the  dangerous  medium  of  a  dog-infested 
watercress  bed.  The  cysticercus  may  be  found  in  almost  any 
region  of  the  body  in  man,  and  occasionally  numbers  of  cysts 
may  be  passed  in  the  faeces  following  the  rupture  of  a  parent 
cyst  into  the  gut.  The  cysts  may  be  quite  small,  translucent, 
round  bodies  not  unlike  grapes.  Their  nature  is  certainly 
determined  by  the  finding  of  barbed  hooklets  or  well-formed 


Fig.  22. — Taenia  Echinococcus.     Scolices  and  Hooklets. 

scolices  within  them,  but  it  is  not  uncommon  to  find  numbers 
of  well -developed  but  sterile  cysts. 

Nemathelminths. — These  are  round,  unsegmented  worms, 
usually  tapering  at  both  ends. 

They  are  divided  into  three  classes  : — 

Class  1,  Nematoda,  commonly  parasitic  in  man. 

Class  2,  Nematomorpha,  not  parasitic  in  man. 

Class  3,  Acanthocephala,  very  rarely  parasitic  in  man. 

Only  the  nematodes  are  considered  here. 

Nematodes.  —  The  nematodes  include  a  considerable 
number  of  different  families,  not  all  of  which  are  parasitic, 
while  others  are  very  commonly  parasitic  in  man. 

Strongyloides  intestinalis  is  a  very  common  intestinal 
parasite  in  tropical  countries.  It  passes  through  a  parasitic 
and  a  free-living  form.  The  adult  parasitic  form  lives  in 
the  mucous  membrane  of  the  small  intestine.  It  is  a  cylin- 
drical worm  2*2  mm.  long  with  a  pointed  tail.  Only  one 
sex,  the  female,  is  found.  The  eggs  are  deposited  in  the 
intestinal-mucous  membrane  and  develop  into  larvae,  which 


THE   PARASITOLOGY   OF    THE   F^CES.       335 

leave  the  host  in  the  faeces.  Here  they  develop  further,  cast 
their  skins,  and  become  sexually  mature.  The  females  are 
1  mm.  long,  and  the  males  shorter.  The  sexes  copulate, 
and  the  female  lays  eggs,  from  which  larvse  are  hatched  which 
develop  into  the  parasitic  form,  and  cannot  reproduce  them- 
selves unless  again  reintroduced  into  the  human  intestine. 
The  larvae  of  the  parasitic  form  are  very  frequently  found  in 
the  stools  of  perfectly  healthy  persons,  and  do  not  appear  to 
produce  any  symptoms. 

FUariid.Ee. — The  varieties  of  these  nematode  worms  occur- 
ring  in   the   blood   have  been  described  in  the  chapter  on 


Fig.  23. — Trichocephalus  Dispar. 
Ovum.     Female.     Natural  Size. 

the  parasitology  of  the  blood.  The  Dracuncidus  medinensis, 
or  guinea-worm,  is  considered  in  the  chapter  on  the  skin. 

Trichotrachelidae. — In  man  only  two  parasitic  species  of 
the  nematode  worms  are  known.  They  are  Trichocephalus 
dispar  and  Trichinella  spiralia. 

Trichocephalus  dispar,  or  the  whip-worm,  is  a  bisexual 
worm  with  a  simple  developmental  history.  The  female  is 
about  50  mm.  long,  and  the  male  is  somewhat  smaller. 
The  anterior  two-thirds  of  the  worm  consists  of  a  thin  filiform 
process.  The  posterior  extremity  of  the  male  is  curved  upon 
itself,  and  ends  in  a  rounded  projection  in  which  the  vas 
deferens  opens.  The  eggs  are  oval,  and  have  a  thick  brown 
capsule  with  an  opening  at  each  end.  The  parasite  is  much 
more  commonly  found  in  the  inhabitants  of  tropical  climates 
than  in  temperate  countries.     No  ill  effect  is  produced  by  the, 


336 


CLINICAL   PATHOLOGY. 


worms.     Infection  takes  place  by  ingestion  of  the  ova,  and  no 
intermediate  host  is  required. 

Trichinella  spiralis  is  viviparous,  and  requires  two  flesh- 
eating  hosts  for  development.  The  females  measure  about 
4  mm.  in  length,  and  the  males  half  that  size.  Both  have 
a  pointed  anterior  extremity,  but  taper  to  it  gradually.  The 
posterior  extremity  of  the  male  has  two  short  caudal  appen- 
dages, between  which  the  cloaca  lies.  Trichinosis  in  man  is 
rare  in  Great  Britain,  but  common  in  Germany  and  America. 
The  usual  host  is  the  pig,  which  may  become  infected  by  eating 
dead  rats.  The  rats  carry  on  the  infection  by  eating  each 
other.  Man  becomes  infected  by  eating  imperfectly-cooked 
pork.  The  larvae  are  encysted  in  the  pork,  and  when  swallowed 
are  set  free  in  the  stomach.     They  become  sexually  mature  in 


Fia.  24. — TTicHnella  Spiralis. 
Larva  Encysted  in  Muscle. 

the  upper  part  of  the  small  intestine,  and  copulate.  The  males 
die,  and  are  passed  in  the  faeces.  The  females  bore  through 
the  gut  wall  into  the  lymphatic  spaces,  where  they  pass  their 
larvae.  The  larvae  pass  into  the  circulation,  and  come  to  rest  in 
striated  muscle.  Here  they  become  encysted  between  the 
muscle  fibres.  The  female  dies  after  a  vigorous  productive 
period  of  about  7  weeks. .  The  infection  is  a  serious  one,  and 
the  migration  of  the  larvae  into  the  muscles  is  accompanied 
by  considerable  disturbance  and  local  pain.  A  well-marked 
eosinophilia  is  present  in  the  blood. 

Strongylidse  —  By  far  the  most  important  of  these  worms 
found  in  man  is  the  ankylostoma  (uncinaria). 

Ankylostoma  duodenale  is  a  nematode  worm  which  has 
a  very  wide  distribution,  and  causes  serious  symptoms.  There 
are  two  distinct  species  of  ankylostoma,  A.  duodenale  and 
A.  americanum.      The  latter  has  not  yet  been  met  with  in 


THE   PABASITOLOGY   OF    THE   F.ECES.       337 

this  country.  In  A.  duodenale  the  female  is  10  to  13  mm. 
long  and  the  male  is  somewhat  shorter.  The  anterior 
extremity  of  each  is  rounded,  and  the  buccal  cavity  is 
guarded  by  4  incurved  spines.  The  posterior  extremity  of  the 
female  is  pointed,  while  that  of  the  male  ends  in  a  mem- 
branous expansion  from  which  2  long  spicules  protrude. 
The  eggs  are  oval,  with  a  very  thin  transparent  and  colour- 
less shell.  Segmentation  of  the  contents  has  commenced  when 
they  reach  the  faeces,  2  or  4  cells  being  visible.  In  A.  ameri- 
canum  the  worms  are  smaller  and  the  buccal  cavity  has  no 
spines.       The   worms    are    extremely   widely   distributed   in 


Fig.  25. — Ankylostoma  Duodenale.  Anterior  and  Posterior  Extremity 
of  Male.  Posterior  Extremity  of  FemaLe.  Ovum.  Female. 
Natural  Size. 


tropical  and  subtropical  countries,  but  in  Great  Britain  are  con- 
fined to  places  where  suitable  climatic  conditions  exist  for  the 
development  of  the  ova.  The  infection  is  found  amongst  workers 
in  the  metalliferous  mines  of  Cornwall,  having  been  intro- 
duced from  foreign  countries.  An  extremely  severe  anaemia, 
accompanied  by  a  well-marked  eosinophilia,  follows  the 
infection.  The  adult  worms  live  in  the  small  intestine  of  man, 
being  firmly  attached  by  their  heads  to  the  mucous  membrane. 
The  ova  pass  out  in  the  stools.  The  larvae  hatch  out  under 
favourable  circumstances  in  about  2  days.  The  full-grown 
larva  is  produced  from  the  ovum  in  about  10  days,  and  after 
moulting  three  or  four  times  becomes  a  sexless  individual  moving 
p.  22 


338 


CLINICAL   PATHOLOGY. 


freely  in  a  chitinous  sheath.  The  ova  are  not  infective ;  the 
full-grown  larvae  are.  Man  becomes  infected  in  one  of  two 
ways  ;  either  the  larvae  are  swallowed,  a  method  which  is 
probably  the  less  common,  or  they  enter  through  the  skin, 
causing  a  local  erythremia  or  urticaria,  then  pass  to  the 
lungs,  causing  bronchial  catarrh,  and  finally  find  their  way 
to  the  stomach.  The  diagnosis  is  made  by  the  finding  of  the 
ova  in  the  faeces.  If  the  live  ova  are  incubated  they  will  be 
found  to  contain  larvae  in  about  24  hours.  The  adult  worms 
are  rarely  seen  in  the  faeces. 

Ascaridae. — The  ascaridae  include  two  members  which  are 
commonly  parasitic  to  man  in  this  country,  namely,  Ascaris 
lumbricoides  and  Oxyuris  vermicular  is. 

Ascaris  lumbricoides  is  the  common  round  worm  of  man, 
and  has  an  extremely  wide  geographical  distribution.     The 

female  is  from  20  to 
40  cm.  long,  and  the 
male  about  two-thirds 
that  length.  The 
heads  of  both  sexes 
have  3  prominent  lips, 
2  ventral  and  1  dorsal. 
The  tail  of  the  male 
worm  is  strongly 
curved  in  a  ventral 
direction,  and  2  fine 
spicules  extrude  from  it.  The  worms  are  commonly  found 
in  pairs  in  the  small  intestine,  and  in  the  great  majority 
of  cases  cause  no  symptoms.  They  occasionally,  however, 
wander  from  the  intestine,  and  may  make  their  way  up  the 
common  bile  duct  into  the  gall  bladder  or  into  the  liver,  and 
more  rarely  have  been  found  in  the  pancreatic  duct.  The  worms 
are  only  from  time  to  time  passed  in  the  faeces,  but  the 
diagnosis  can  readily  be  made  as  a  rule  by  finding  the  ova, 
which  have  a  characteristic  appearance.  The  ova  are  fairly 
large,  measuring  "05  by  *07  mm., and  are  more  round  than  oval. 
The  un  segmented  ovum  has  a  thick,  transparent,  colourless  shell, 
which  it  does  not  completely  fill.  The  shell  is  nearly  always 
coated  with  a  rough,  granular  albuminous  material  of  a  brown 
colour.  The  unfertilised  ova  contain  retractile  globules.  The 
embryos  develop  in  a  moist  atmosphere  at  room  temperature 


Fig.  26. — Ascaris  Lurnbricoid.es.  Posterior 
Extremity  of  Male.  Head  Enlarged  and 
Natural  Size.     Ovum. 


THE    PARASITOLOGY   OF    THE    FAECES. 


339 


in  from  30  to  40  days,  and  can  remain  alive  for  long  periods. 
The  life-history  is  simple  ;  the  ova  containing  the  embryos 
are  ingested,  the  capsules  are  dissolved,  and  the  free  larvae 
reach  maturity  in  the  intestine  in  about  5  weeks. 

Somewhat  similar,  but  smaller,  ascaridae  infest  dogs  and 
cats,  and  are  occasionally  found  in  man. 

Oxyuris  vermicularis  is  vulgarly  known  as  the  thread- 
worm, and  is  a  common  parasite  of  man  and  particularly  of 
children  of  the  poorer  classes.  The  female  is  about  10  mm. 
long,  and  the  male  about  half  that  length.  The  tail  of 
the  female  is  straight  and  pointed  ;  that  of  the  male  is  curved 
and  rounded.  The  two  sexes  live  and  copulate  in  the  small 
intestine.  The  fertilised  females  leave  the  males  and  travel 
downwards  to  form  large  congregations  in  the  caecum,  appendix, 


Fig.  27. — Oxyuris  Vermicularis.     Male.     Female  and  Natural 
Size.     Ovum  with  Embryo. 

and  ascending  colon,  where  they  reside  until  the  ova  are  nearly 
mature.  The  females  then  set  out  on  their  travels  again, 
pass  down  the  rectum  and  appear  at  the  anus.  The  ova  are 
deposited  on  the  mucous  membrane  and  skin  of  the  anus  and 
perineum.  The  wanderings  of  the  worms,  the  irritation 
the  ova  and  of  the  larvae,  which  may  escape  from  them, 
cause  considerable  perineal  itching.  The  child  scratches  its 
perineum,  and  conveys  the  ova  to  its  mouth  or  nose  or  to  the 
face  of  its  neighbour.  The  ova  loose  their  capsules  in  the 
stomach,  and  the  freed  embryos  reach  maturity  in  about  a  fort- 
night. Fertilised  females  containing  mature  eggs  are  often 
swept  out  with  the  faeces,  and  the  diagnosis  is  made  by  detecting 
them  in  the  stools.  The  free  ova  are  rarely  found.  The  ova  are 
of  about  the  same  size  and  shape  as  those  of  ankylostoma,  and 
are  enclosed  in  a  thin  capsule.     They  are,  however,  distinctly 

22—2 


340  CLINICAL   PATHOLOGY. 

flattened  on  one  side,  and  at  the  time  of  passage  contain  a 
well-developed  embryo. 

Amoebae. — Amcebic  dysentery  is  practically  confined  to 
tropical  or  almost  tropical  countries,  and  consequently  has  a 
much  narrower  geographical  range  than  bacillary  dysentery. 
Amoebic  dysentery  is  common  in  India  and  Africa,  and  in  many 
parts  of  these  countries  is  the  form  of  dysentery  most  fre- 
quently met  with.  Very  rare  examples  of  tropical  abscess  of  the 
liver,  possibly  due  to  the  Amoeba  dysenterica,  have  been  met 
with  in  patients  who  have  never  been  out  of  Great  Britain. 
The  diagnosis  of  amoebic  dysentery  rests  with  the  detection 
of  the  causative  organism  in  the  stools.  In  the  large  majority 
of  cases  the  organisms  are  extremely  numerous  in  the  acute 
stage  and  readily  found.  All  that  is  necessary  is  to  place  a  drop 
of  the  blood-stained  "  mucus  "on  a  slide  with  a  cover-slip  over 
it  and  examine  with  a  ^-inch  objective.  A  warm-stage  and 
hanging-drop  preparation  is  not  essential,  but  the  amoebic 
movements  are  more  certainly  seen  by  this  method.  The 
stool  should  always  be  examined  within  an  hour  or  two  of 
being  passed,  and  large  doses  of  ipecacuanha  should  be  with- 
held until  the  examination  is  made.  On  a  cold  day  the 
ordinary  slide  preparation  should  be  warmed,  and  in  any  case 
it  should  be  examined  immediately  after  it  has  been  made.  If 
the  amoebae  are  few  in  number  a  second  piece  of  the  mucus 
should  be  picked  out  and  mounted  in  a  drop  of  1  per  cent, 
watery  methylene  blue  with  a  cover-slip  over  it.  The  advan- 
tages of  this  method  are  that  the  leucocytes  and  epithelial  cells 
are  immediately  stained  blue,  but  the  amoebae  resist  taking  the 
stain,  while  retaining  their  mobility  for  a  considerable  time. 
The  clear,  retractile  bodies  of  the  amoebae  stand  out  very 
prominently  on  the  blue  background,  and  can  be  detected  with 
a  f-inch  objective,  the  higher  power  being  reserved  for 
confirmation  of  the  diagnosis. 

Amoebae  of  more  than  one  variety  are  to  be  met  with  in  the 
fasces.  One  is  a  perfectly  harmless  and  normal  inhabitant  of 
the  intestinal  tract,  and  has  to  be  distinguished  from  the 
dysenteric  organism.  The  harmless  amoeba  is  known  as  the 
Amoeba  or  Entamoeba  coli :  the  dysenteric  organism  is  called 
the  Entamoeba  histolytica.  The  Entamoeba  coli,  which  is  found 
in  the  upper  part  of  the  large  gut,  is  of  comparatively  small 
size,  and  has  a  well-defined  nucleus  and  little  clear  ectoplasm. 


THE   PARASITOLOGY  OF   THE   F.ECES.       341 

The  Amoeba  histolytica  is  a  large  amoeba  measuring  from  25 
to  35  fju  in  diameter,  and  has  an  abundant,  clear,  refractile  ecto- 
plasm, and  a  poorly-defined  nucleus  which  stains  badly  with 
the  ordinary  dyes. 

The  distinction  between  the  two  amoebae  is  not  of  very 
great  practical  importance  in  a  dysenteric  district,  since  the 
harmless  amoeba  is  very  rarely  met  with,  and  if  amoebae  are 
found  in  considerable  numbers  in  any  stools  the  case  may 
safely  be  diagnosed  as  amoebic  dysentery. 

Bacteriology. — The  faeces  are  normally  so  infested  with 
bacteria  that  the  difficulty  is,  not  to  obtain  a  growth  in  culture 
media,  but  to  isolate  the  pathogenic  organisms  from  the  non- 
pathogenic and  to  obtain  them  in  pure  culture. 

Some  departure  from  the  ordinary  routine  bacteriological 
methods  is  necessary,  and  some  clinical  advice  is  particularly 
required  as  to  the  class  of  organism  to  be  sought  for.  The 
detection  of  organisms  known  to  be  pathogenic  and  to  be 
normally  absent  from  the  intestinal  tract,  such  as  the  cholera 
vibrio  or  the  typhoid  bacillus,  is  of  diagnostic  significance. 
The  detection  of  organisms  of  atypical  cultural  characters  and 
of  doubtful  pathogenicity  should  be  amplified  so  far  as  possible 
by  other  methods,  such  as  the  investigation  of  the  agglutinat- 
ing action  of  the  patient's  serum  upon  them,  and  the  findings, 
unless  strongly  supported  by  such  means,  must  be  accepted 
with  reserve.  The  detection  of  bacteria  known  to  be  found  in 
the  gut  in  health,  such  as  the  bacillus  coli,  has  no  diagnostic 
significance. 

-  Film  preparations  of  the  faeces  commonly  give  little  bacterio- 
logical information,  but  they  should  be  made  in  the  case  of 
very  liquid  stools  in  order  to  gain  an  idea  of  the  type  of  the 
prevalent  organism.  In  cases  of  cholera  the  vibrio  may  be 
present  in  enormous  numbers  in  the  films.  The  cultural 
investigation  necessarily  varies  with  the  type  of  organism 
looked  for,  but  in  the  majority  of  cases  the  following  method 
will  be  found  useful  as  a  routine  : — 

The  faeces  should  be  passed  into  an  ordinary  clean  (not 
carbolised)  bed-pan,  and  should  be  examined  as  soon  as  possible 
after  they  have  been  passed. 

In  an  ordinary  broth  culture  tube  take  10  to  12  loops  of  the 
faeces  with  a  sterile  platinum  wire. 

Shake  the  broth  tube  thoroughly,  without  letting  the  fluid 


342  CLINICAL   PATHOLOGY. 

splash  against  the  wool  plug,  and  stand  the  tube  aside  for  a 
few  moments  to  allow  the  solid  particles  to  settle. 

Take  a  loop  of  the  supernatant  fluid  and  plate  on  an  agar 
plate. 

Incubate  the  broth  tube  for  from  -I  to  6  hours  at  37°  C. 

Take  a  loop  of  the  supernatant  broth  and  plate  it  out  on  3 
MacConkey  plates,  without  recharging  the  loop. 

Incubate  the  4  plates  till  the  next  morning. 

Examine  the  agar  plate  for  colonies  of  the  staphylococcal 
and  streptococcal  type. 

Examine  the  MacConkey  plates  for  yellow  colonies  in 
particular. 

Take  sub-cultures  of  the  suspected  colonies  in  the  usual 
way. 

The  points  to  be  aimed  at  in  this  process  are  to  obtain  plate 
cultures  which  are  neither  hopelessly  overgrown  nor  contain 
merely  one  or  two  colonies,  but  which  show  a  fail*  number  of 
discrete  colonies  on  the  majority  of  the  plates. 

The  more  important  pathogenic  organisms  to  be  met  with 
are  the  following  : — 

The  typhoid  bacillus. — The  bacilli  can  be  isolated  from 
the  fasces  by  the  above  method.  The  yellowish  colonies  are 
picked  out  from  the  MacConkey  plates  and  two  or  three  of 
them  subcultured  into  broth.  The  broth  cultures  are  then 
subcultured  into  the  appropriate  media.  If  bacilli  are  obtained 
which  culturally  resemble  the  typhoid  bacillus  it  is  wise  to  test 
them  with  the  serum  from  a  known  case  of  typhoid  fever  as  well 
as  with  the  serum  from  the  patient. 

Paratyphoid  bacilli. — These  organisms  are  looked  for  in 
exactly  the  same  manner,  and  in  all  cases  the  agglutinating 
action  of  the  patient's  serum  upon  them  should  be  investi- 
gated. The  sera  of  animals  inoculated  against  the  various 
organisms  can  be  obtained  from  a  few  reliable  sources,  and 
should  also  be  tested  upon  them. 

The  dysentery  bacillus. — This  organism  is  investigated 
in  the  same  manner.  Either  the  Shiga-Kruse  or  the  Flexner 
type  may  be  found,  and  serum  reactions  should  always  be 
investigated.  The  bacilli  may  be  met  with  in  cases  of  dysen- 
tery and  colitis  in  this  country,  and  have  been  isolated  in 
some  epidemics  of  infantile  summer  diarrhoea  in  America. 

A  bacillus  of  the  same  class,  which  has  been  associated  with 


THE   PARASITOLOGY  OF    THE    FAECES.        343 

British  epidemics  of  infantile  diarrhoea,  has  the  cultural 
peculiarity  of  failing  to  ferment  mannite,  and  is  known  as 
Morgan's  No.  1  bacillus. 

The  cholera  vibrio. — The  recognition  of  the  cholera  vibrio 
is  a  comparatively  simple  matter  in  an  epidemic,  or  in  districts 
where  cholera  is  known  to  be  rife. 

Sporadic  cases  of  cholera  are  to  be  diagnosed  with  extreme 
caution,  since  the  differentiation  between  the  genuine  cholera 
vibrio  and  other  vibrios  which  may  occasionally  be  present  in 
the  gut  is  a  matter  of  considerable  difficulty.  The  vibrios 
other  than  the  cholera  vibrio  will  be  mentioned  later. 

The  examination  of  the  faeces  should  be  conducted  as 
follows : — 

(1)  Prepare  a  thin  film  of  the  faeces,  and  stain  it  with  carbol- 
thionin.  In  an  acute  case  the  organisms  are  usually  present 
in  considerable  numbers,  and  tend  to  appear  in  groups,  all  the 
members  of  which  lie  with  their  long  axes  in  the  same  direc- 
tion, as  trout  lie  in  a  stream. 

(2)  Put  several  loops  of  the  faeces  into  broth,  and  incubate  for 
about  4  hours. 

Plate  from  the  broth  on  to  3  gelatin  plates,  and  incubate  at 
from  18°  to  20°  C.  for  24  hours. 

Examine  the  plates  for  colonies  of  the  vibrio.  These 
colonies  are  white  with  jagged  outlines,  and  have  the  appear- 
ance of  powdered  glass  on  the  surface  of  the  plate.  Film 
preparations  from  them  show  the  vibrio. 

(3)  Subculture  from  the  gelatin  plate  into  broth,  on  to  agar 
slopes,  and  in  gelatin  stab  cultures.  Continue  the  incubation 
of  the  gelatin  plate. 

After  incubation  for  from  24  to  48  hours  of  the  broth  culture 
add  to  it  a  few  drops  of  pure  sulphuric  acid.  A  rose-pink 
colour  of  nitroso-indole  develops  in  the  medium. 

The  agar  slope  culture  shows  a  yellowish  irregular  slimy 
growth. 

The  gelatin  stab  culture  shows  a  white  streak  of  growth 
along  the  track  of  the  inoculating  wire,  and  at  the  surface  of 
the  medium  a  funnel-shaped  depression  of  commencing 
liquefaction. 

The  colonies  on  the  gelatin  plate  slowly  darken,  and  lique- 
faction takes  place  in  the  medium. 

(4)  With   the   pure   culture    on    the    agar   slope   make    a 


344  CLINICAL   PATHOLOGY. 

suspension  of  the  vibrios  in  order  to  test  the  lytic  action  of  an 
immune  serum  upon  them  (Chapter  VIII.,  page  110). 

The  crucial  test  of  the  true  vibrio  rests  with  the  specfic 
action  of  the  lytic  serum  upon  it.  It  is  not  a  practical 
necessity  to  perform  this  test  in  cases  met  with  in  an  epidemic, 
but  the  notification  of  a  case  of  cholera  in  a  previously  cholera- 
free  district  is  a  serious  matter,  demanding  that  every  reason- 
able bacteriological  precaution  should  be  taken  to  ensure  an 
accurate  diagnosis. 

Among  the  organisms  liable  to  be  confounded  with  the 
vibrio  of  Asiatic  cholera  are  the  following  : — 

The  vibrio  of  cholera  nostras  described  by  Finkler  and 
Prior  may  be  found  in  considerable  numbers  in  the  stools  of 
patients  suffering  from  acute  diarrhoea  and  vomiting  with 
collapse,  such  as  may  occur  in  temperate  climates  in  the 
summer  months.  In  the  majority  of  such  sporadic  cases, 
however,  vibrios  in  the  stools  are  either  very  scanty  or 
absent.  The  Finkler-Prior  vibrio  is  longer  and  broader  than 
Koch's  vibrio,  and  on  gelatin  plate  culture  forms  round 
colonies  with  sharply-cut  edges.     Gelatin  is  liquefied  rapidly. 

Similar  vibrios,  which  may  be  found  in  the  stools  of  healthy 
persons  or  patients  with  intestinal  affections,  have  been  isolated 
from  cheese  and  from  the  water  supplies  of  towns.  Some  of 
these  vibrios  closely  resemble  the  cholera  vibrio  both  on 
morphological  and  cultural  grounds,  and  can  only  certainly  be 
distinguished  by  serum  tests. 

Inoculation  of  the  cholera  vibrio  into  the  peritoneal  cavity 
of  guinea-pigs  induces  a  typical  toxic  effect.  Other  vibrios 
are  non-pathogenic  to  animals. 

Tubercle  bacillus. — This  organism  may  be  present  in 
the  faeces  in  large  numbers  in  cases  of  tuberculous  enteritis, 
and  in  such  cases  pus  is  present  in  addition,  and  often  blood. 
The  bacilli  are  present  in  small  numbers  in  the  case  of 
persons  with  pulmonary  tuberculosis,  and  particularly  of 
children,  who  are  more  likely  to  swallow  the  sputum. 

The  faeces  should  be  treated  with  antiformin  and  the  bacilli 
looked  for  in  the  ordinary  way.  Occasionally  the  addition  of 
antiformin  to  faeces  produces  a  brilliant  red  colour ;  in  such 
cases  the  patients  have  been  found  to  be  taking  "  purgen  "  or 
phenol-phthalein,  which  turns  red  on  the  addition  of  alkaline 
antiformin. 


THE    PARASITOLOGY   OF    THE   F.ECES.       345 

Other  organisms. — Among  other  of  the  commoner 
organisms  to  be  met  with  in  the  fasces  are  staphylococci  and 
streptococci ;  their  significance  is  uncertain.  Cocci  are  com- 
monly present  in  the  normal  fasces,  and  the  streptococci  met 
with  are  as  a  rule  of  very  low  pathogenicity  for  animals.  In 
cases  of  ulcerative  colitis  the  organisms  commonly  suspected 
and  looked  for  as  the  causative  agents  are  members  of  the  coli- 
typhoid  group,  and  it  is  quite  possible  that  coccal  infection 
may  in  some  cases  be  overlooked. 

The  cultural  examination  of  colitis  is  aided  by  the  passage 
of  the  sigmoidoscope.  After  thorough  irrigation  of  the  colon 
with  sterile  tap  water  a  minute  portion  of  the  infected  mucous 
membrane  can  be  safely  removed  by  a  skilled  operator  and 
transferred  to  a  culture  tube.  By  such  means  streptococci 
may  be  found  in  pure  culture  in  a  small  percentage  of  cases,  and 
a  vaccine  prepared  from  the  organism  may  be  beneficial. 

The  isolation  of  cocci  from  fasces  by  the  ordinary  methods 
may  be  difficult  and  can  have  no  particular  significance. 


SECTION  VI. 

THE   EYE   AND    SKIN. 

CHAPTEE  XXIV. 

The  Eye  and  Conjunctival  Sac — The  Skin. 


CHAPTER   XXIV. 

the  eye  and  conjunctival  sac — the  skin. 
The  Eye  and  Conjunctival  Sac. 

The  cytology  of  the  conjunctival  sac  does  not  materially 
differ  from  that  of  other  parts  of  the  body.  In  the  more 
chronic  infections  of  the  conjunctiva  large  epithelial  cells  are 
commonly  met  with,  and  in  the  acute  inflammations  the 
ordinary  polynuclear  cells  of  suppuration.  In  the  rare  con- 
dition known  as  "  spring  catarrh "  the  exudate  consists 
almost  entirely  of  eosinophil  cells. 

The  bacteriology  of  the  eye  is  of  considerable  importance, 
and  in  a  variety  of  affections  much  assistance  is  obtained 
from  a  careful  bacteriological  investigation. 

The  normal  conjunctiva,  being  exposed  to  air  contami- 
nation, is  rarely  sterile,  and  some  acquaintance  with  the 
organisms  present  in  health  is  necessary.  The  bacillus 
known  to  ophthalmologists  as  the  xerosis  bacillus  is  the 
organism  most  frequently  met  with,  and  is  present  in  the 
majority  of  cases  examined.  The  xerosis  bacillus  is  no  longer 
believed  to  play  any  essential  part  in  the  production  of  the 
condition  the  name  of  which  it  bears.  It  is  a  diphtheroid 
organism,  and  will  be  subsequently  referred  bo  under  that 
name.  The  diphtheroid  bacilli  met  with  in  the  eye  are  of 
more  than  one  variety,  but  the  great  majority  of  them  belong 
to  a  type  which  grows  readily  on  the  ordinary  solid  media, 
such  as  agar  or  blood  serum,  but  merely  maintains  its 
existence  in  broth  and  other  liquid  media.  It  does  not 
acidify  litmus  dextrose  broth.  Next  to  the  diphtheroid  bacilli 
a  white  staphylococcus  of  low  virulerice  is  the  organism 
most  frequently  met  with.  Exceptionally,  more  virulent 
organisms,  such  as  staphylococcus  aureus,  streptococcus 
pyogenes  and  the  pneumococcus  may  be  cultivated  from 
the  apparently  normal  conjunctiva. 

The  occasional  finding  of    virulent  organisms    in    normal 


348  CLINICAL   PATHOLOGY. 

cases  is  of  considerable  importance,  and  it  is  a  reasonable 
precaution  to  make  a  bacteriological  examination  in  all  cases 
before  conducting  such  an  operation  as  that  of  cataract 
extraction.  It  is  an  essential  precaution  if  any  inflammatory 
condition  of  the  lids  or  conjunctiva  is  present,  however 
mild. 

Conjunctivitis- — The  commonest  and  most  widely-spread 
variety  of  acute  conjunctivitis  is  that  due  to  the  Koch-Weeks 
bacillus.  The  disease  has  a  short  incubation  period  of  about 
24  hours,  is  extremely  contagious,  begins  as  a  rule  in  one  eye 
and  almost  invariably  spreads  to  the  other,  is  associated  with 
redness  and  swelling  of  the  lids  and  conjunctivae,  and  runs 
a  variable  course,  often  lasting  from  2  to  4  weeks.  The 
diagnosis  can  be  sufficiently  confirmed  by  film  preparations. 
The  minute,  slender  Gram- negative  bacilli  closely  resemble 
in  appearance  the  influenza  bacillus,  from  which  there  is  no 
practical  necessity  to  identify  them.  They  are  of  very 
variable  length,  and  tend  to  appear  in  small  clusters  in  the 
films.  They  are  both  intra-  and  extra-  cellular.  It  is  fortunate 
that  the  bacillus  can  be  thus  identified  in  film  preparations, 
since  their  growth  on  culture  media  is  extremely  difficult  to 
obtain.  Special  media  containing  some  form  of  blood  serum 
are  required,  and  the  organism  is  frequently  outgrown  by 
diphtheroid  bacilli  or  other  bacteria. 

Diplo-bacillary  conjunctivitis. — The  causative  organism 
of  this  disease  is  known  as  the  Morax-Axenfeld  bacillus. 
The  condition  is  widely  distributed,  and  is  more  chronic  than  the 
Koch-Weeks'  infection.  It  is  infectious,  almost  always  affects 
both  eyes,  and  is  associated  with  a  characteristic  redness  of  the 
angles  of  the  palpebral  fissure.  The  infection  may  in  untreated 
cases  last  for  years.  A  nasal  catarrh  is  sometimes  present 
in  addition,  and  the  causative  organism  may  be  found  in  the 
nasal  discharge.  In  films  made  from  the  pus  the  bacilli  are 
found  as  stout  rods  of  moderate  length  with  rounded  ends, 
the  great  majority  of  them  being  in  pairs  and  outside  the 
cells.  Occasional  chains  are  present.  The  occurrence  of 
Gram-negative  bacilli  of  this  nature  in  film  preparations  is 
sufficient  to  establish  a  diagnosis.  A  growth  of  the  bacilli  in 
the  form  of  small,  translucent  colonies  on  serum  agar  can 
often  be  obtained. 

Gonorrhoea!  conjunctivitis  has  been  already  referred  to 


EYE   AND   CONJUNCTIVAL    SAC— SKIN.         349 

in  the  description  of  the  gonococcus.  The  causative  organism 
is  as  a  rule  numerous  in  the  conjunctival  secretion,  and  the 
condition  is  readily  recognised  in  film  preparations. 

Trachoma. — The  cause  of  this  disease  is  unknown,  and 
numerous  parasites,  subsequently  discredited,  have  been  from 
time  to  time  described. 

Other  varieties  of  conjunctivitis. — A  purulent  con- 
junctivitis may  be  set  up  by  any  of  the  ordinary  pyogenic 
bacteria,  and  the  investigation  of  the  exudate  should  be 
conducted  on  the  ordinary  lines.  In  no  case  should  a 
diagnosis  of  the  organisms  be  made  from  film  preparations 
alone.  Mixed  infections  are  not  infrequent.  In  a  series  of 
investigations  dealing  with  this  class  of  infection  the  staphy- 
lococcus albus  was  found  twenty-eight  times,  staphylo- 
coccus aureus  twenty-seven  times,  a  streptococcus  ten 
times,  an  intermediate  staphylococcus  three  times,  the 
diphtheria  bacillus  twice,  and  an  unclassified  coccus  once. 
Diphtheroid  bacilli  were  also  present  in  about  60  per  cent, 
of  the  cases.  In  addition  to  these  organisms  the  pneumo- 
COCCUS  may  exceptionally  be  found  as  the  cause  of  a  primary 
conjunctivitis,  and  it  has  been  described  as  a  common  cause 
owing  to  the  improper  identification  of  the  organism  by  film 
preparations  only.  Epidemics  of  conjunctivitis  due  to  the 
pneumococcus  have  been  recorded.  The  diphtheria  bacillus  is 
a  rare  cause  of  conjunctivitis,  being  usually  met  with  in 
children  and  in  association  with  a  nasal  discharge  containing 
the  same  organism.  The  diphtheria  bacillus  should  be  identified 
in  this  situation,  not  only  by  its  morphological  and  cultured 
characters,  but  also  by  animal  inoculation.  Diphtheritic 
infection  of  the  conjunctiva  is  associated,  as  elsewhere,  with  mem- 
brane formation,  and  is  readily  amenable  to  serum  treatment. 

Membranous  conjunctivitis  is  less  commonly  due  to 
the  diphtheria  bacillus  than  to  other  organisms,  of  which  the 
most  important  is  the  Streptococcus  -pyogenes.  Streptococcal 
infection  of  the  conjunctiva  is  the  most  virulent  type  met 
with,  and  a  small  percentage  of  cases  proceed  rapidly  to 
panophthalmitis  in  spite  of  all  treatment.  Less  commonly 
staphylococci  may  produce  a  membranous  conjunctivitis. 

The  lids. — The  bacteriology  of  the  lids  is  practically 
identical  with  that  of  the  skin,  and  all  the  commoner  varieties 
of  inflammation  associated  with  the  pyogenic  cocci  are  met 


350  CLINICAL   PATHOLOGY. 

with.  Molluscum  contagiosum  is  occasionally  encountered 
here,  as  elsewhere,  on  the  exposed  surfaces. 

The  lachrymal  sac. — Inflammations  of  the  sac  are  set  up 

by  the  ordinary  pyogenic  organisms,  of  which  a  streptococcus 
is  perhaps  the  most  common.  Here  again  pneumococci  may 
be  present,  but  have  been  described  more  frequently  than  is 
probably  correct  owing  to  an  undue  reliance  upon  film  prepara- 
tions. The  micrococcus  catarrhalis  is  another  organism 
occasionally  associated  with  this  condition. 

The  cornea. — The  main  organism  concerned  in  the  pro- 
duction of  the  serpiginous  corneal  ulcer  is  the  pneumo- 
COCCUS.  The  infection  is,  however,  not  a  truly  specific  one, 
and  other  organisms,  such  as  the  staphylococci  and  strepto- 
cocci, may  be  present  in  pure  culture.  The  identification  of 
the  pneumococcus  has  been  in  some  cases  a  matter  of  con- 
siderable doubt,  and  when  cultures  have  been  taken  the 
organism  has  been  depicted  as  growing  in  long  chains  of 
30  members  or  more,  a  condition  practically  not  met  with 
among  pneumococci  from  other  sources.  Bacilli  of  the 
Colon  group,  B.  proteus  and  B.  pyocyaneus,  are  occasionally 
found  in  corneal  ulceration,  as  well  as  in  exceptional  cases  of 
conjunctivitis  without  corneal  infiltration.  Aspergillosis  of 
the  cornea  has  also  been  recorded  on  numerous  occasions. 

The  chronic  infective  granulomata. — Tuberculosis, 
leprosy  and  syphilis  may  each  effect  the  eye.  The  methods 
of  recognising  the  causative  organism  are  the  same  as  those 
adopted  for  these  diseases  in  other  parts  of  the  body. 

Endogenous  infections. — Metastatic  abscess  in  the 
eye  may  arise  in  the  course  of  a  general  infection  produced 
by  any  of  the  pyogenic  organisms.  An  interesting  variety  of 
metastatic  ophthalmitis  occurs  in  epidemics  of  cerebro-spinal 
meningitis,  and  a  similar  condition,  producing  a  disease  known 
as  " pseudo-glioma,"  has  recently  been  demonstrated  to  be 
caused  by  the  meningococcus,  and  to  occur  in  cases 
with  no  history  of  meningeal  symptoms.  The  coccus  has 
been  recovered  from  the  interior  of  the  eye  after  excision. 

The  orbit.— The  bacteriology  of  acute  inflammatory  con- 
ditions of  the  orbit  calls  for  no  particular  description. 
Infection  of  the  orbit  arises  by  direct  extension  from  adjacent 
parts  in  the  great  majority  of  cases.  The  most  important 
sources  of  infection  are  the  accessory  sinuses  of  the  nose. 


EYE   AND   CONJUNCTIVAL   SAC— SKIN.        351 

The    Skin. 

There  is  scarcely  any  pathological  investigation  which  may 
not  from  time  to  time  be  required  for  patients  whose  main 
complaint  is  of  some  skin  lesion.  The  following  are  among 
some  of  the  changes  more  particularly  associated  with  diseases 
of  the  skin  : — 

The  blood. — An  eosinophilia,  often  of  marked  degree,  is 
frequently  associated  with  many  widespread  dermal  lesions. 
In  the  specific  fevers  associated  with  skin  eruptions,  and 
particularly  in  small-pox,  chicken-pox,  and  scarlet  fever,  a 
considerable  eosinophilia  is  the  rule.  In  the  two  former 
diseases  the  eosinophils  diminish  in  number  and  finally  dis- 
appear when  the  bullae  suppurate.  Among  other  diseases 
associated  with  a  vesicular  eruption  dermatitis  herpetiformis 
is  almost  constantly  accompanied  by  a  considerable  eosino- 
philia. In  films  made  from  the  bullae  in  these  cases,  as  well 
as  in  small-pox  and  chicken-pox,  numerous  leucocytes  are 
present,  and  the  great  majority  of  them  are  eosinophils.  In 
pemphigus  chronicus,  on  the  other  hand,  eosinophils  are 
absent  from  the  blebs  and  there  is  no  eosinophilia  in  the 
blood.  The  cytological  character  of  dermal  exudates  is  thus 
of  some  assistance  in  differential  diagnosis.  Secondary 
infection  of  skin  vesicles,  however,  very  readily  occurs,  and  it 
is  necessary  to  examine  the  fluid  as  soon  as  possible  after  the 
vesicle  has  appeared.  Cases  are  not  infrequently  met  with 
in  which  vesicles  have  been  deliberately  produced  by  the 
patient  either  by  the  aid  of  blistering  fluid  or  some  cruder 
device.  In  these  mechanical  effusions  the  great  majority  of 
cells  present  are  of  the  epithelial  type,  and  the  presence  of 
such  cells  is  strongly  suggestive  of  an  artificial  lesion. 

Leukaemia  is  very  rarely  associated  with  considerable 
leuksemic  infiltrations  of  the  skin.  These  infiltrations  have, 
however,  on  occasion  been  so  considerable  as  to  merit  the 
name  of  "  tumours,"  and  the  patients  have  first  come  under  the 
observation  of  a  skin  clinic.  Rare  as  the  condition  is,  it  is 
advisable  in  all  cases  of  multiple  skin  tumours  of  doubtful 
origin  to  make  an  examination  of  the  blood.  The  blood 
changes  present  may  be  either  those  of  myeloid  leukseinia  or 
of  the  lymphatic  variety. 

Pernicious  anaemia  is  nearly  always  accompanied  by  a 


352  CLINICAL   PATHOLOGY. 

lemon-yellow  colour  of  the  skin,  but  in  so  far  as  the  patient  is 
concerned  this  is  a  matter  of  secondary  importance.  A  rare 
condition  may  be  met  with  known  as  hemochromatosis,  in 
which  the  patient  seeks  advice  upon  the  discoloration  of  his 
skin.  This  may  be  of  a  deep  brown  or  almost  black  colour,  and 
the  change  may  have  taken  place  in  a  few  weeks  or  months. 
The  discoloration  is  associated  with  an  abnormal  destruc- 
tion of  red  cells  and  the  liberation  of  pigment  from  them. 
The  blood  as  a  rule  shows  all  the  changes  typical  of  advanced 
pernicious  anaemia. 

Syphilis. — In  some  continental  clinics  all  cases  of  syphilis 
are  treated  by  the  dermatologist,  and  in  this  country  numerous 
patients  present  themselves  for  examination  principally  for  a 
skin  syphilide.  Consequently  an  examination  of  the  Wasser- 
mann  reaction  in  the  serum  is  frequently  required,  and  should 
be  performed  in  all  cases  of  syphilitic  disease  with  skin  lesions 
as  well  as  for  patients  with  rashes  of  a  doubtful  nature.  The 
spirochete  should  also  be  looked  for  in  the  primary  sore,  and 
may  be  found  in  the  condylomata  and  other  secondary  lesions. 

The  parasitology  of  skin  diseases.  —  The  skin  is 
peculiarly  liable  to  infection  both  by  animal  and  vegetable 
parasites,  and  a  complete  description  of  all  such  organisms 
as  may  be  found  in  the  skin  can  only  be  given  in  a  book 
devoted  to  dermatology.  A  brief  account  only  of  the  more 
important  skin  parasites  is  given  here.  The  majority  of  them 
have  been  mentioned  in  the  section  on  bacteriology. 

Animal  parasites— Acarus  scabiei  is  the  parasite  of 
scabies,  or  vulgarly  "itch."  The  diagnosis  of  this  common 
condition  should  be  confirmed  by  the  very  simple  examination 
needed  to  demonstrate  the  causative  parasite.  On  the  skin, 
and  usually  between  the  fingers,  is  seen  a  thin,  greyish  black 
raised  line  about  \  inch  long,  forming  the  burrow  produced  by 
the  female  acarus  as  it  travels  from  the  surface  along  the 
skin.  The  burrow  is  dissected  out  with  a  surgical  needle,  and 
at  the  extremity  furthest  from  the  point  of  entrance  is  found  a 
small  black  speck  just  visible  to  the  naked  eye,  and  evident 
under  a  hand  magnifying  glass  as  the  female  acarus.  At 
intervals  between  the  female  and  the  surface  are  found  the 
ova.  The  male  acarus  does  not  leave  the  surface  of  the  skin, 
and  is  in  consequence  rarely  observed.  The  female  acarus, 
examined  with  a  low   power  of  the  microscope,  is  seen  as  a 


EYE   AND   CONJUNCTIVAL    SAC- SKIN.        353 

somewhat  rounded  oval  body  with  8  limbs.  The  anterior  4 
limbs  are  armed  with  suckers,  the  posterior  4  with  bristles. 
The  male  acarus  is  similar  but  smaller.  The  ova  may  be 
detected  by  dissecting  out  a  portion  of  the  burrow  and  mount- 
ing it  in  saline  or  weak  potash  on  a  slide  with  a  cover-slip  over 
it.  The  oval  eggs  with  a  more  or  less  developed  contained 
embryo  must  be  distinguished  from  the  epithelial  cells  of  the 
skin  with  their  angular  shape  and  central  nucleus. 

Pediculi. — These  loathsome-looking  parasites  are  remark- 
ably particular  in  their  habitat.  Different  varieties  affect 
different  parts  of  the  human  body,  and  practically  never  trans- 
gress upon  each  other's  domains.  Moreover  the  pediculi  of 
some  animals  will  not  pass  to  other  species  of  animals :  for 
example,  the  common  body  lice  of  dogs  do  not  attack  man. 
The  special  body  lice  of  man  are  named  according  to  their 
distribution,  Pediculus  corporis,  Pediculus  capitis,  and  Pedicu- 
lus pubis. 

Pediculus  corporis  infests  the  trunk  and  the  body  clothing. 
It  is  the  largest  of  the  human  pediculi,  being  about  3  mm. 
long  and  readily  visible  to  the  naked  eye.  The  ova  are 
laid  upon  the  hairs  or  the  clothing.  Pedicidus  corporis,  as 
is  the  case  with  the  other  pediculi,  has  6  legs  armed  with 
short  claws.     It  attacks  adults  more  commonly  than  children. 

Pediculus  capitis  is  confined  to  the  hairy  scalp.  It  has  a 
similar  shape  to  Pediculus  corporis,  but  is  not  so  long.  The 
numerous  white  ova  or  "  nits  "  are  attached  to  the  hairs  and 
form  very  conspicuous  objects,  while  the  adult  parasites  can 
readily  be  seen  on  close  inspection  moving  among  the  hair 
roots.  The  infection  is  extremely  common,  especially  among 
children  with  long  hair.  Adults,  particularly  women  of  the 
lower  classes,  are  by  no  means  exempt. 

Pediculus  pubis  is  shorter  and  considerably  stouter  than 
either  of  the  two  former  species.  It  is  about  1^  mm. 
long.  Its  shape  has  earned  it  the  euphonious  name  of 
"  crab  louse."  The  ova  are  brown,  and  are  attached  to  the 
hairs  in  the  same  manner  as  those  of  Pedicidus  capitis.  Its 
range  is  almost  entirely  confined  to  the  pubic  hairs,  but  the 
hairs  of  the  axillae  and  the  eye-lashes  may  be  exceptionally 
infected.  The  spread  of  this  parasite  is  usually  by  sexual 
intercourse. 

Leptus  autumnalis  is  an  extremely  common  larva  which 

p.  23 


354  CLINICAL   PATHOLOGY. 

attacks  the  human  skin.  Its  vulgar  name  is  "harvest  bug." 
In  many  country  districts  the  parasite  is  very  numerous  on 
grass  lands  in  July  and  August,  and  attacks  through  thin 
clothing  any  part  of  the  human  body  which  may  come  in 
contact  with  the  ground.  The  minute  parasite  buries  its 
head  in  the  skin. 

The  common  flea  and  the  bed  bug  are  objects  sufficiently 
familiar  to  need  no  particular  description.  The  reaction  of 
the  individual  attacked  is  very  variable,  and  the  extreme 
swelling  in  a  susceptible  person  may  be  very  puzzling  if  the 
central  puncture  left  by  the  bite  be  not  recognised.  There 
seems  to  be  both  a  natural  and  an  acquired  immunity  to  the 
poison,  and  persons  who  make  a  practice  of  harbouring  these 
creatures  may  show  little  swelling  and  no  evidence  of 
irritation. 

Dracunculus. — The  guinea-worm  is  a  common  source  of 
disease  in  the  tropics,  and,  since  the  sojourn  of  the  parasite 
in  the  human  body  is  about  one  year,  occasional  instances 
of  infection  are  met  with  in  this  country.  The  cycle  of 
development  is  by  way  of  a  small  water  Crustacea  to  the 
human  being,  in  whom  the  adult  worm  reaches  maturity.  The 
male  worm  fertilises  the  female  and  disappears.  The  fertile 
female  migrates  into  the  tissues,  and  leaves  the  body  by 
piercing  the  skin  in  the  most  convenient  position  for  deposit- 
ing her  embryos  in  water.  The  skin  is  thus  most  commonly 
punctured  in  the  neighbourhood  of  the  foot  or,  if  the  host  be 
a  water-carrier,  in  the  back.  The  worm  is  about  the  thick- 
ness of  a  piece  of  string  and  some  30  inches  long.  When 
the  worm  comes  to  the  surface  it  is  best  removed  by  the  time- 
honoured  expedient  of  winding  it  on  a  stick  a  few  inches  at  a 
time.  The  practice  of  injecting  the  worm  with  perchloride  of 
mercury  is  less  certain,  since  the  dead  worm  is  rendered  more 
brittle  and  may  have  to  be  dissected  out  along  its  tortuous 
bed,  or  allowed  to  suppurate  out. 

Fungi. — Microsporon  Audouini  is  a  common  cause  of 
ringworm  of  the  scalp  and  body.  It  is  peculiar  to  the  human 
race  and  almost  confined  to  children.  The  spores  are  closely 
attached  around  the  shaft  of  a  hair,  and  the  short  mycelial 
threads  are  scanty.  Cultures  on  maltose  agar  produce  round, 
white,  downy  colonies  with  a  central  tuft. 

Trichophyton  megalosporon  endothrix  produces  ring- 


EYE   AND   CONJUNCTIVAL    SAC- SKIN.         355 

worm  of  the  scalp  and  body,  and  rarely  of  the  nails.  It  is 
peculiar  to  the  human  race.  The  spores  are  arranged  in 
chains  within  the  hair.  The  colonies  on  maltose  agar  may 
be  white,  greyish  yellow,  primrose,  or  violet. 

Trichophyton  megalosporon  ectothrix  is  properly  a 
parasite  of  ungulates,  dogs,  cats,  and  birds,  but  is  transmissible 
to  man.  Ringworm  of  the  body,  beard,  and  nails  is  produced, 
the  scalp  being  only  occasionally  attacked.  The  spores  are 
arranged  in  chains,  and  the  mycelium,  which  may  be  abun- 
dant, is  jointed.  There  are  numerous  varieties  of  this  fungus, 
which  can  be  distinguished  by  the  colour  and  appearance  of 
their  colonies. 

Achorion  Schoenleinii  is  the  commonest  of  the  favus 
fungi,  and  affects  the  scalp,  skin,  and  nails.  It  has  a  branched 
mycelium  with  numerous  large  spores.  Cultures  are  brownish 
yellow,  with  a  ridged  surface. 

Microsporon  furfur  infects  the  hairy  layer  of  the 
epidermis  of  uncleanly  persons.  The  disease  produced  is 
known  as  pityriasis,  or  tinea  versicolor.  The  parasite  has  an 
abundant  branching  mycelium,  interspersed  with  clumps  of 
spores. 

Microsporon  minutissimum  affects  moist  regions  of  the 
body,  producing  a  lesion  known  as  erythrasma.  The  fungus 
consists  of  an  abundant  mycelium  composed  of  very  fine  long, 
unbranched  threads.     Spores  are  scanty. 

The  diagnosis  of  these  fungoid  diseases  should  always  be 
confirmed  by  the  microscope.  The  simplest  method  of 
demonstrating  the  mycelium  and  spores  is  to  remove  an  affected 
hair,  or  to  scrape  off  the  epithelial  scales  of  the  skin  or  the 
parings  of  infected  nails,  and  mount  them  in  liquor  potassae 
on  a  slide  beneath  a  cover-slip. 

Other  fungi  are  rarely  present  in  skin  lesions  met  with  in 
this  country.  Actinomycosis  of  the  skin  is  very  unusual, 
and  the  skin  is  practically  only  affected  by  extension  from  the 
deeper  structures.  The  organism  is  to  be  sought  in  the  yellow 
granules  of  the  pus.  Madura  foot,  or  mycetoma,  another 
cutaneous  affection,  is  met  with  in  India  and  East  Africa,  and  is 
caused  by  a  variety  of  fungoid  organisms.  Blastomyces,  or 
yeast  fungi,  have  been  shown  to  produce  a  nodular  infectious 
disease  of  the  skin,  and  the  organisms  may  become  dissemi- 
nated through  the  system.     The  sporotrichia  are  organisms 

23—2 


356  CLINICAL   PATHOLOGY. 

possessed  with  a  regular  septate  mycelium  and  spore-bearing 
branches.  They  produce  a  granulomatous  condition  of  the 
skin,  simulating  the  lesions  of  tuberculosis  and  syphilis.  The 
fungus  grows  slowly  on  appropriate  media,  and  the  serum  of 
affected  persons  acquires  agglutinins  for  a  suspension  of  the 
spores  obtained  from  a  culture.  The  condition  has,  compara- 
tively recently,  attracted  considerable  attention. 

Bacteria. — The  Normal  Skin. — The  normal  skin,  like  the 
normal  conjunctiva,  being  an  exposed  surface,  is  in  conse- 
quence rarely  sterile.  The  organisms  to  be  met  with  on 
almost  any  skin  and  in  almost  any  part  of  the  body  are  white 
staphylococci,  sarcinae,  and  diphtheroid  bacilli.  These 
organisms  are  under  ordinary  circumstances  non-pathogenic. 
It  is  possible  that  they  play  some  part  in  the  normal  meta- 
bolism of  the  skin,  and  that  in  diseased  conditions  they  may 
even  become  pathogenic.  The  number  of  these  organisms 
present  on  the  skin  of  different  individuals  varies  considerably, 
and  they  are  a  common  source  of  contamination  in  culture 
tubes,  whether  derived  from  the  defective  technique  of  the 
bacteriologist  or  from  the  skin  of  the  patient.  The  diphtheroid 
bacilli  and  sarcinse  are  readily  recognised,  and  their  presence 
in  films  or  in  culture  tubes  may  always  be  regarded  as  evidence 
of  contamination.  The  staphylococci  of  the  normal  skin  are 
almost  always  represented  by  a  white  variety  which  is  very 
inactive  in  culture  media,  growing  readily  but  producing  little 
change. 

Less  commonly  more  virulent  organisms  may  be  grown 
from  the  normal  skin,  and  the  majority  of  pathogenic  species 
can  on  occasion  be  obtained.  Staphylococcus  aureus,  or 
citreus,  and  the  streptococci  are  the  most  important 
organisms  met  with,  and  were  of  particular  importance  to  the 
surgeon  before  the  introduction  of  rubber  operating  gloves. 
They  are  still  of  importance  if  present  on  the  skin  of  the 
patient.  Virulent  organisms  can  maintain  their  existence 
on  the  skin  without  producing  any  harmful  results  upon 
their  host.  They  may  exist  for  considerable  periods  in 
spite  of  attack  by  all  the  ordinary  methods  of  cleanliness, 
including  antiseptics.  The  action  of  antiseptics  applied  to  the 
skin  rarely  succeeds  in  destroying  all  bacterial  growth  without 
injury  to  the  epidermis.  A  minute  trace  of  a  powerful  anti- 
septic, however,  will  inhibit  or  delay  the  subsequent  growth  of 


EYE   AND   CONJUNCTIVAL   SAC— SKIN.        357 

organisms,  and  in  cultures  taken  from  a  focus  which  has  been 
exposed  to  antiseptic  action  it  is  common  to  find  the  visible 
growth  of  a  freely-multiplying  species  delayed  by  two  or  three 
clays. 

Streptococcal  infections  of  the  skin  are  common,  and  the 
most  notorious  infection  produced  is  erysipelas.  Erysipelas 
proper  is  an  acute  inflammation  of  the  dermis  caused  in  the 
great  majority  of  cases  by  the  Streptococcus  pyogenes.  The 
cultural  investigation  is  not  always  successful,  since  material 
may  not  be  available  for  cultural  purposes.  The  organisms 
spread  along  the  superficial  layers  of  the  skin,  but  cultures 
made  from  recent  bullse  are  in  a  considerable  percentage  of 
cases  sterile.  If  cellulitis  is  present  in  addition,  and  incisions 
are  made,  the  streptococci  can  be  obtained  in  pure  culture.  The 
local  lesion  in  erysipelas  may  provide  the  causative  organism, 
but  in  cases  of  facial  erysipelas  particularly  the  local  lesion 
may  not  be  obvious,  or  may  be  situated  in  the  nasal  cavity 
and  consequently  form  the  habitat  of  numerous  other 
bacteria. 

Impetigo  contagiosa  is  a  primary  infectious  disease  most 
often  met  with  in  children,  and  produced  by  the  Streptococcus 
pyogenes.  The  secondary  impetigo  produced  by  scratching 
in  patients  affected  with  scabies,  pediculosis,  or  other  irritative 
lesions  may  be  streptococcal,  or  more  commonly  staphy- 
lococcal, in  origin ;  mixed  infections  are,  however,  frequent. 
The  follicular  impetigo  of  the  hair  follicles  is  essentially  a 
staphylococcal  disease.  Pemphigus  neonatorum  is  usually 
associated  with  a  septic  condition  of  the  umbilical  cord  stump, 
and  is  a  disease  attended  with  a  high  mortality.  The  causa- 
tive organism  may  be  a  streptococcus  or  a  staphylococcus. 

Staphylococcal  infections  of  the  skin  are,  as  might  be 
expected,  extremely  common,  and  very  varied  lesions  are  pro- 
duced. Follicular  impetigo  of  the  skin  and  of  the  scalp  is 
a  frequent  infection,  particularly  among  ill-cared-for  children. 
The  condition  may  be  transmitted  by  one  child  to  another,  is 
liable  to  relapse,  and  often  runs  a  prolonged  course.  The 
causative  organism  is  nearly  always  the  Staphylococcus  aureus, 
but  mixed  infections  are  not  infrequent.  The  lesions  are 
readily  inoculated  by  the  patient  from  one  part  of  the  body  to 
another.  Staphylococcal  inflammations  of  the  eyelids  or  con- 
junctivae  are    often   produced    by  auto-inoculation    from    an 


358  CLINICAL   PATHOLOGY. 

impetiginous  focus  on  the  finger  or  the  face.  In  refractory 
cases  benefit  is  often  derived  from  vaccine  treatment.  It  is 
preferable  that  an  autogenous  vaccine  should  be  given  in  this 
and  in  all  other  staphylococcal  infections  of  the  skin. 

Boils  are  sequels  of  follicular  impetigo,  and  due  to  the  same 
organisms.  They  are  prone  to  occur  in  the  debilitated  as  well 
as  among  those  in  particularly  vigorous  health.  Carbuncles 
are  a  more  serious  lesion  produced  by  staphylococci,  of  which 
Staphylococcus  aureus  is  the  most  frequent  agent.  Vaccine 
treatment  is  often  of  value  as  an  aid  to  surgery,  but  should 
never  be  given  to  the  exclusion  of  thorough  surgical  inter- 
ference. The  majority  of  the  cutaneous  infections  produced 
by  staphylococci  tend  to  run  a  chronic  course  and  to  recur, 
and  attempted  immunisation  with  vaccines  is  reasonable  in 
most  cases. 

Multiple  subcutaneous  abscesses  due  to  staphylococcal 
infection  are  sometimes  met  with,  and  are  not  uncommon  as  a 
sequel  to  prolonged  fevers.  Typhoid  fever  is  fairly  often 
associated  in  the  post-febrile  stage  with  the  appearance  of 
numerous  and  widely-distributed  subcutaneous  abscesses.  The 
Staphylococcus  aureus  or  albus  is  nearly  always  obtained  in 
pure  culture,  and  I  have  never  found  the  typhoid  bacillus  in 
these  lesions.  Subcutaneous  abscesses  may  result  from  a 
general  blood  infection,  and  be  associated  with  pus  formation 
in  other  parts  of  the  body ;  but  in  the  majority  of  cases  the 
patient's  general  condition  is  good,  and  the  abscesses  appear 
to  result  rather  from  the  local  atrophic  condition  of  the  skin 
following  fever  and  the  prolonged  stay  in  bed.  The  production 
of  bed  sores  is  a  similar  process  in  which  pressure  plays  a 
prominent  part. 

Whitlow  is  often  of  staphylococcal  origin,  and  when  accom- 
panied by  a  spreading  lymphangitis  is  practically  always 
due  to  the  Staphylococcus  aureus. 

Barber's  rash,  or  sycosis,  is  one  of  numerous  local  skin  in- 
fections, many  of  which  are  provided  with  special  names  and 
the  majority  of  which  are  due  to  staphylococci.  Seborrhcea 
may  be  produced  by  a  variety  of  organisms,  including 
staphylococci,  and  in  view  of  the  extraordinarily  septic  habits 
of  many  barbers,  even  in  the  most  highly-gilded  saloons,  it 
is  remarkable  that  more  violent  infections  are  not  frequently 
transferred  from  one   victim    to  another.      Fortunately    the 


EYE   AND   CONJUNCTIVAL    SAC— SKIN.        359 

highly-priced  "  tonics  "  applied  after  the  operation  usually 
contain  some  cheap  and  useful  antiseptic. 

Acne  vulgaris  is  an  inflammation  of  the  sebaceous  glands, 
commencing  as  a  rule  at  puberty  and  rarely  lasting  beyond 
the  twenty-fifth  year.  The  causative  organism  is  a  small 
Gram-positive  one  allied  to  the  diphtheroid  group,  and  a 
variety  of  them  probably  exist.  They  may  be  obtained  from 
the  comedones  before  suppuration  occurs,  and  less  commonly 
after  it  has  taken  place.  Suppuration  is  usually  the  result  of 
a  mixed  infection  by  the  acne  bacillus  and  the  Staphylococcus 
albus.  The  result  of  vaccine  treatment  in  these  cases  is  very 
difficult  to  judge,  owing  to  the  natural  variability  of  the  disease 
and  its  tendency  to  sudden  cessation  apart  from  treatment.  A 
very  guarded  prognosis  should  be  given  before  commencing 
vaccine  treatment,  and  it  is  wise  to  warn  the  patient  that  the 
scars  of  old  lesions  will  not  be  affected  by  the  vaccine.  Old- 
standing  cases  with  deep  scarring  are  perhaps  least  often 
affected  by  vaccine  treatment,  while  cases  with  comedones  and 
no  pus  formation  are  the  most  favourable.  In  nonsuppura- 
tive cases  doses  of  5  to  10  million  of  the  bacilli  should  be 
given,  and  in  the  suppurative  cases  the  best  results  may  be 
obtained  with  a  coccal  vaccine,  or  with  a  mixed  vaccine  of  cocci 
and  bacilli. 

Anthrax  is  a  rare  infection  occurring  among  hide  porters, 
wool  sorters,  and  butchers.  An  account  of  the  anthrax  pustule 
and  of  the  bacillus  has  been  given  in  a  previous  chapter. 

Other  pyogenic  organisms  which  may  affect  the  skin 
are  numerous ;  but  the  majority  of  such  infections  are  not 
primarily  dermal  and  spread  to  the  skin  by  extension  from 
the  deeper  tissues. 

Tuberculosis  of  the  skin  assumes  many  clinical  forms,  and 
on  a  pathological  basis  may  be  divided  into  at  least  two 
varieties.  In  one  form  the  tubercle  bacilli  are  present  in  the 
lesion,  although  as  a  rule  in  very  small  numbers.  The 
bacilli  are  in  a  considerable  percentage  of  cases  of  the  bovine 
type,  and  the  lesions  which  they  produce  have  to  be  recognised 
mainly  from  their  clinical  features.  Material  is  not  ordinarily 
available  for  bacteriological  examination,  and  the  diagnosis 
has  to  be  confirmed  when  necessary  by  removal  of  a  portion 
of  skin.  The  histological  evidence  of  tuberculosis  can 
sometimes   be    confirmed   by    detecting    the     bacilli   in    the 


360  CLINICAL   PATHOLOGY. 

sections,  but  the  organisms  are  commonly  very  scanty,  and 
often  proof  can  only  be  completed  by  inoculation  of  portions 
of  tissue  into  guinea-pigs.  Diagnostic  injections  of  old 
tuberculin,  or  Von  Pirquet's  reaction,  are  nearly  always  posi- 
tive, and  a  negative  result  to  either  test  is  strong  evidence 
against  tuberculosis. 

In  the  other  class  of  tuberculous  infections  the  bacillus 
appears  to  be  entirely  absent  from  the  lesion,  and  to  such 
conditions  the  term  "  tuberculides  "  is  applied.  It  is  supposed 
that  the  affections  may  be  caused  by  the  toxins  of  the  bacillus 
which  have  been  absorbed  from  a  distant  focus. 

Leprosy  is  characteristically  associated  with  the  formation 
of  granulomata  in  the  skin,  and  the  diagnosis  can  nearly 
always  be  confirmed  by  the  examination  of  swabs  taken  from 
the  nasal  secretion.  The  lepra  bacilli  are  often  numerous  in 
this  situation. 

Syphilis  and  the  means  of  detecting  the  Spirochceta  pallida, 
together  with  the  importance  of  the  Wasserman  reaction, 
have  already  been  mentioned. 

Another  disease  affecting  the  skin  and  produced  by 
spirochetes  is 

Yaws.  This  disease  has  some  clinical  features  in  common 
with  syphilis,  and  is  produced  by  a  spirochete  extremely  like 
the  Spirocliceta  pallida. 


SECTION  VII. 

THE   KESPIRATOBY   TEACT. 

CHAPTEE  XXV. 

The  Nose — The  Sputum. 


CHAPTER  XXV. 

the  nose — the  sputum. 
The  Nose. 

The  examination  of  the  nasal  secretion  is  mainly  bacterio- 
logical, and  since  cultures  taken  from  the  nasal  cavity  are 
practically  never  sterile,  the  results  of  bacterial  examination 
in  disease  must  be  critically  considered.  The  methods  of 
bacterial  investigation  do  not  materially  differ  from  those 
previously  described. 

It  is  advisable  as  a  routine  to  use  a  cotton-wool  swab  similar 
to  that  employed  for  diphtheria  cultures,  and  to  plate  direct 
from  it  on  to  two  agar  plates.  A  further  culture  may  also  be 
made  into  broth,  and  film  preparations  should  be  made  in 
addition  in  all  cases. 

The  following  are  among  the  affections  which  may  especially 
require  investigation : — 

Nasal  catarrh. — The  usual  attack  of  nasal  catarrh  is 
commonly  left  to  run  its  course  with  or  without  the  aid  of 
domestic  remedies.  Some  persons  are  so  unfortunate  as  to 
suffer  from  repeated  attacks  at  short  intervals,  and  particu- 
larly during  the  autumn  and  winter  months.  Vaccine 
treatment  has  been  very  largely  employed  to  meet  such  cases. 
Provided  there  is  no  local  nasal  condition  such  as  can  be 
rectified  by  the  surgeon,  and  the  patient  is  seriously  embar- 
rassed by  the  attacks,  it  is  reasonable  to  attempt  to  isolate  the 
causative  organism  and  prepare  a  vaccine  from  it.  Excep- 
tional patients  appear  to  derive  actual  benefit :  others  obtain 
mental  comfort  from  the  administration  of  a  hypodermic 
injection  :  many  are  unaffected.  On  the  whole  the  benefit  to 
be  expected  is  so  uncertain  that  it  is  not  justifiable  to  urge  a 
course  of  vaccine  therapy,  or  even  to  advise  it,  except  as  a 
last  resort. 

The  organisms  which  may  be  met  with  include  the  micro- 
coccus   catarrhalis,    streptococci,    staphylococci,    and 


THE    NOSE -THE    SPUTUM.  363 

less  commonly  pneumococci  and  Friedlander's  pneumo- 

baciUus.  The  vaccine  is  preferably  made  from  the  pre- 
dominant and  most  virulent  organism.  The  selection  of  the 
causative  bacterium  is  partly  a  matter  of  chance,  and  a  mixed 
vaccine  may  be  employed. 

Hay  fever. — A  standardised  vaccine  of  pollen  toxin  has 
been  given  for  this  condition.  Affected  patients  give  an 
ophthalmic  reaction  to  the  pollen  similar  to  the  Calmette 
reaction  to  tuberculin.  The  method  is  still  on  its  trial  and  is 
not  described  here. 

Diphtheria. — Nasal  diphtheria  is  not  infrequently  met 
with  in  children,  and  the  bacteriological  examination  is  con- 
ducted in  the  same  manner  as  in  tonsilar  diphtheria. 
Harmless  diphtheroid  bacilli,  however,  are  more  commonly 
met  with  in  the  nose  than  in  the  throat,  and  in  cases  of 
clinical  doubt  it  is  advisable  to  inoculate  a  guinea-pig  with  a 
culture  of  the  suspected  organism. 

Leprosy. — The  examination  of  the  nasal  secretion  in  cases 
of  leprosy  has  already  been  considered.  The  bacilli  are  almost 
invariably  present  in  the  nose  in  considerable  numbers  and 
in  the  early  stages  of  the  disease.  There  may  be  little  actual 
discharge  noticeable  to  the  patient,  and  the  secretion  is  often 
thick,  crusted,  and  difficult  to  manipulate. 

The  Sputum. 

General  examination  of  the  sputum. — Naked-eye 
observations. — The  amount  of  the  sputum  is  of  importance 
in  certain  diseases,  and  in  particular  in  cases  of  bronchiectasis 
and  pulmonary  tuberculosis.  The  amount  may  vary  from  a  few 
cubic  centimetres  in  the  early  morning  to  as  much  as  a  litre 
in  the  24  hours. 

The  odour  is  practically  inoffensive  in  the  majority  of 
affections,  but  may  be  quite  overpowering.  Bronchiectastic 
sputum  is  nearly  always  offensive,  as  is  also  that  from  an 
abscess  of  the  lung.  If  gangrene  of  the  lung  is  present  the 
odour  is  indescribable. 

The  colour  is  whitish  in  early  and  mild  catarrhal  cases. 
It  becomes  yellow  with  advancing  suppuration.  It  is  red  if 
blood  is  present.  Blackish  particles,  visible  to  the  naked  eye, 
are  nearly  always  present,  and  are  due  to  inspired  atmospheric 
carbon. 


364  CLINICAL   PATHOLOGY. 

The  consistence  and  general  appearance  is  some  guide  to 
the  condition.  The  sputum  in  pneumonia  is  particularly 
viscid,  and  in  tuberculosis  of  the  lung  the  sputum  may  have 
a  "  nummular "  character.  Fibrinous  casts,  spirals,  and 
shreds  of  solid  tissue  may  in  certain  conditions  be  visible  to 
the  naked  eye. 

Microscopical  examination.  —  The  sputum  can  be 
examined  microscojjically  both  in  the  fresh  state,  by  squeezing 
a  portion  between  a  slide  and  a  cover-slip,  and  in  stained  film 
preparations. 

The  stained  films  show  in  the  majority  of  cases  large  epithe- 
lial cells  and  leucocytes  of  the  polynuclear  variety  in  varying 
proportions,  fibrinous  strands,  and  large  numbers  of  organisms. 
The  structures  to  be  examined  for  are  the  following  : — 

The  cells. — Epithelial  cells  are  nearly  always  present,  but 
if  they  form  the  great  majority  of  the  cells  in  a  film  the 
"  sputum  "  probably  comes  mainly  from  the  mouth  and  upper 
air  passages.  Tubercle  bacilli  are  rarely  found  in  such  speci- 
mens, which  are  liable  to  be  produced  by  a  patient  to  order 
and  to  be  examined,  with  the  misleading  result  that  tubercle 
bacilli  are  stated  to  be  absent. 

Pus  cells  are  present  in  all  sputa  whether  the  underlying 
condition  is  tuberculous  or  not. 

Eosinophils  are  not  recognised  in  carbol-thionin  or  methy- 
lene blue  preparations  ;  they  must  be  specially  stained  for, 
and  are  well  seen  in  thin  films  treated  with  Leishman's  stain 
in  the  ordinary  way.  Eosinophils  may  form  the  predominant 
cell  in  cases  of  genuine  spasmodic  asthma. 

Red  blood  corpuscles  may  be  recognised  in  the  fresh  speci- 
mens and  in  the  stained  films.  They  stain  a  greenish  colour 
with  carbol-thionin,  but  are  best  fixed  and  stained  by  one  of 
the  blood  stains. 

Elastic  fibres.- — Before  the  discovery  of  the  tubercle 
bacillus  the  presence  of  elastic  fibres  in  the  sputum  was 
regarded  as  of  great  diagnostic  importance.  The  fibres  are 
now  rarely  looked  for,  but  their  presence  in  sputum  is  of 
significance,  since  they  indicate  that  there  has  been  actual 
destruction  of  lung  tissue.  Elastic  fibres  in  small  numbers 
may  find  their  way  into  the  sputum  from  the  food,  and  it  is 
advisable  to  instruct  the  patient  to  cleanse  his  mouth 
thoroughly  before  obtaining  a  sample  of  sputum. 


THE    NOSE— THE    SPUTUM.  365 

Elastic  fibres  are  found  in  tuberculosis,  bronchiectasis,  and 
pulmonary  abscess.  They  are  occasionally  met  with  in  lobar 
pneumonia  apart  from  abscess  formation.  In  actual  gangrene 
of  the  lung  the  fibres  are  rarely  found,  probably  because  they 
have  been  dissolved  locally  by  ferment  action. 

Single  elastic  fibres  are  difficult  to  detect  and  are  of  little 
diagnostic  importance,  since  they  may  have  been  introduced 
in  the  food,  unless  very  particular  care  has  been  taken. 
Elastic  fibres  occurring  in  bundles  which  display  an  alveolar 
arrangement  are  more  readily  detected,  and  certainly  come 
from  the  lung. 

The  fibres  vary  in  size,  and  have  a  wavy  outline  and  double 
contour.  If  they  are  present  in  considerable  numbers  it  is  only 
necessary  to  mix  a  little  sputum  on  a  slide  with  10  per  cent, 
caustic  potash,  and  to  spread  it  out  under  a  cover-slip.  The 
fibres  are  more  resistant  to  the  potash  than  the  other  con- 
stituents of  the  sputum,  and  stand  out  as  curved  refractile 
threads. 

If  the  elastic  fibres  are  few  in  number  add  to  some  sputum 
in  a  test  tube  an  equal  part  of  10  per  cent,  caustic  potash. 
Boil  until  the  sputum  is  dissolved.  Mix  the  solution  with 
four  times  its  own  volume  of  water.  Allow  the  mixture 
to  stand  for  24  hours,  and  examine  the  deposit  for  elastic 
fibres. 

Curschmann's  spirals. — These  spiral  bodies  are  found  in 
great  numbers  in  the  sputum  of  cases  of  spasmodic  asthma. 
They  are  not  found  in  asthmatic  cases  of  old  standing  in 
whom  advanced  emphysema  and  bronchitis  have  occurred. 
The  sputum  in  such  cases  is  of  the  ordinary  bronchial  type.  The 
spirals  are  present  in  the  sputum  which  immediately  follows 
a  true  spasmodic  attack.  They  are  not  confined  to  asthma,  but 
may  be  occasionally  observed  in  cases  of  acute  pulmonary 
tuberculosis. 

The  spirals  are  visible  to  the  naked  eye,  and  appear  as  white 
twisted  tenacious  bodies  in  the  sputum.  Examined  under  the 
microscope  they  are  seen  to  consist  of  a  coarse  central  thread 
round  which  is  wound  a  twisted  meshwork  of  delicate  fibrils. 
When  straightened  out  the  spirals  may  be  2  or  3  inches  long. 
The  fibrils  appear  to  consist  of  a  central  thread  of  fibrin 
around  which  tendrils  of  mucin  are  wound.  The  spirals  are 
often  embedded  in  epithelial  cells,  and  may  contain  in  addition 


366  CLINICAL   PATHOLOGY. 

Charcot-Leyden  crystals.  Their  source  of  origin  is  probably 
the  smaller  bronchioles. 

Charcot-Leyden  crystals. — These  are  colourless,  elon- 
gated, and  sharply -pointed  octahedral  crystals.  They  are 
insoluble  in  water,  alcohol  or  ether,  and  soluble  in  acids  and 
alkalies.  They  are  frequently  found  in  the  sputum,  particularly 
after  standing,  of  asthmatic  patients,  but  are  not  diagnostic  of 
this  disease,  and  probably  are  of  no  particular  significance. 

Fibrinous  casts. — Small  fibrinous  casts  of  the  finer 
bronchioles  are  occasionally  detected  in  the  sputum  in  lobar 
pneumonia,  broncho-pneumonia,  and  rarely  in  bronchitis. 
They  are  whitish  in  colour,  and  of  moderately  firm  consistence. 
Large  fibrinous  casts  are  practically  confined  to  the  very  rare 
condition  known  as  fibrinous,  or  chronic  plastic,  bronchitis. 
The  fibrinous  coagulum  consists  of  a  branched  stem  with 
numerous  sub-divisions,  resembling  the  leafless  branch  of  a 
tree.  Almost  the  complete  cast  of  a  bronchial  system  may 
occasionally  be  expectorated. 

The  sputum  in  various  diseases. — Bronchitis  in  the 
earlier  stages  is  accompanied  by  a  whitish,  viscid,  and  scanty 
sputum,  which  later  becomes  yellow,  copious,  and  obviously 
purulent.  The  sputum  is  rarely  "  nummular,"  but  cannot 
be  distinguished  by  its  appearance  from  that  of  tuberculous 
cases. 

Bronchiectasis. — The  sputum  is  copious,  and  is  usually 
brought  up  in  large  quantities  at  a  time,  with  considerable 
intervals  between  the  attacks  of  expectoration.  It  is  as  a  rule 
comparatively  fluid,  and  almost  invariably  has  a  highly- 
offensive  odour. 

Pneumonia. — At  the  onset  of  lobar  pneumonia  the  sputum 
is  very  scanty  or  absent,  and  in  exceptional  cases  there  may 
be  very  little  sputum  throughout  the  course  of  the  disease. 
Later  the  sputum  becomes  abundant  and  is  characteristically 
tenacious.  Owing  to  the  intimate  admixture  of  the  blood  and 
exudation  in  the  pulmonary  tissues  the  sputum  is  more  or  less 
evenly  blood-stained,  and  is  commonly  described  as  "  rusty  "  in 
appearance. 

Pulmonary  tuberculosis. — In  the  majority  of  cases  there 
is  little  in  the  appearance  of  the  sputum  by  which  tuberculous 
cases  can  be  distinguished  from  other  pulmonary  conditions. 
The  presence  of  blood  in  the  sputum,  but  not  intimately  mixed 


THE    NOSE— THE    SPUTUM.  367 

with  it  as  in  lobar  pneumonia,  is  always  suggestive  of  tuber- 
culosis. The  blood  is  bright  red  as  a  rule,  and  may  be  in 
large  amount,  or  the  sputum  may  be  streaked  with  blood. 
Nummular  sputum  is  also  indicative  of  tubercle  of  the  lung 
with  cavity  formation.  The  nummular  character  is  best  seen 
by  floating  the  sputum  in  water,  when  the  round,  more  or 
less  flattened,  discs  of  muco-pus  separate  out  and  finally  sink 
to  the  bottom.  The  only  valuable  evidence  of  tuberculous 
sputum  is  the  finding  of  the  tubercle  bacillus.  The  bacilli 
are  to  be  found  in  the  very  great  majority  of  all  cases  of 
pulmonary  tuberculosis  associated  with  a  considerable  degree 
of  expectoration. 

Influenza  is  associated  in  the  early  stages  with  sputum  of 
the  ordinary  bronchial  character,  and  in  the  later  stages  often 
becomes  extremely  profuse  and  very  tenacious.  The  bacilli 
can  be  detected  as  a  rule  in  ordinary  film  preparations. 

Asthma. — In  cases  of  spasmodic  asthma  the  sputum  com- 
mences as  the  dyspnoea  passes  off,  and  small  characteristic 
pellets  are  coughed  up.  The  pellets  contain  eosinophil  cells 
in  large  numbers,  Curschmann's  spirals,  and  often  Charcot- 
Leyden  crystals. 

Abscess. — In  abscess  of  the  lung  the  sputum  may  consist 
of  pure  pus  with  practically  no  mucoid  admixture.  In  cases 
of  any  standing  the  smell  is  always  offensive.  The  abscess 
may  come  from  the  lung  itself  or  from  the  pleural  cavity  after 
rupture  of  an  empyema  into  the  lung.  The  material 
expectorated  may  be  indistinguishable  from  the  contents  of 
a  bronchiectatic  cavity.  In  all  such  cases  numerous  fine, 
long  bacilli  are  present  and  are  probably  responsible  for  the 
odour. 

A  tropical  abscess  of  the  liver  may  rupture  into  the  lung 
and  give  to  the  sputum  an  appearance  of  having  been  mixed 
with  anchovy  sauce.  Amoebae  may  be  detected  in  the 
sputum. 

Gangrene  is  associated  with  sputum  of  a  green  colour 
and  extremely  offensive  odour.  Gangrene  of  the  lung  may 
follow  an  injury,  and  is  the  rarest  sequel  of  lobar  pneumonia. 

Malignant  disease  of  the  lung  is  usually  associated  with 
haemorrhagic  sputum.  The  expectoration  has  been  pleasantly 
compared  to  red  currant  jelly. 

Malignant  disease    of   the  oesophagus   is   very   commonly 


368  CLINICAL   PATHOLOGY. 

associated  with  a  very  profuse,  pale,  watery,  and  tenacious 
secretion.  The  occurrence  of  this  material  in  the  sputum  pot 
is  strong  evidence  of  a  malignant,  as  opposed  to  a  spasmodic, 
stricture  of  the  oesophagus. 

(Edema  of  the  lung  is  accompanied  by  a  copious  white, 
frothy  sputum.  "When  blood  is  present  in  addition  the 
expectoration  is  commonly  likened  to  prune  juice. 

Infarction  of  the  lung  is  accompanied  by  a  bright  red 
sputum  intimately  mixed  with  froth. 

Pneumoconiosis  results  in  a  brownish  black  sputum. 
The  colour  is  due  to  particles  of  carbon  in  "  anthracosis,"  or 
of  sulphide  of  iron  in  "  siderosis,"  or  to  lime  dust  in  "  stone- 
mason's lung." 

The  parasitology  of  the  sputum. — Infection  of  the 
lungs  by  the  higher  animal  parasites  is  not  common.  In 
hydatid  disease  of  the  lungs  or  pleura  the  characteristic 
hooklets  can  sometimes  be  found  in  the  sputum,  and  occasional 
small  cysts  may  be  coughed  up  entire.  In  cases  of  endemic 
haemoptysis  met  with  in  China  and  other  parts  of  eastern  Asia 
the  ova  of  the  Paragonimus  westermani  are  to  be  looked  for 
in  the  sputum.  The  parasite  has  been  described  in  the  section 
on  the  faeces. 

The  amoebae  of  dysentery,  after  rupture  of  a  liver  abscess 
into  the  lung,  are  looked  for  in  the  sputum  in  exactly  the 
same  manner  as  in  the  faeces  (p.  340).  Failure  to  find  the 
amoebae  is  not  uncommon.  The  organisms  are  more  likely  to 
be  met  with  2  or  3  days  after  the  rupture  of  the  abscess  has 
taken  place. 

Bacteriology  of  the  sputum. — In  almost  every  pul- 
monary affection  the  sputum  swarms  with  a  variety  of 
organisms,  and,  apart  from  the  detection  of  the  tubercle 
bacillus,  the  recognition  of  the  causative  bacterium  is  usually  a 
matter  of  considerable  uncertainty. 

The  following  procedure  may  be  followed  for  the  routine 
bacteriological  examination  of  the  sputum  : — 

(1)  The  mouth  should  be  cleansed  as  far  as  possible  before 
the  material  is  expectorated,  but  strong  antiseptics  must  be 
avoided  in  the  cleansing. 

(2)  A  sterile  test  tube  fitted  with  a  cotton-wool  cork  and  a 
sterile  glass  filter  funnel  is  provided,  and  the  patient  is  directed 
to  remove  the  cork  from  the  tube,  to  insert  the  funnel,  and  to 


THE   NOSE— THE    SPUTUM.  369 

expectorate  down  the  funnel  into  the  tube.     The  funnel  is 
then  removed  and  the  tube  re-corked. 

(3)  Elaborate  methods  of  washing,  filtering,  and  teasing  the 
sputum  are  in  use ;  but  perfectly  satisfactory  results  are 
obtained  by  siruply  taking  a  loop  of  the  sputum  in  a  sterile 
platinum  wire,  and  plating  it  out  on  two  agar  plates  without 
re-charging  the  loop.  Discrete  colonies  are  almost  invariably 
obtained  in  this  way. 

A  broth  culture  is  preferably  put  up  as  a  control  at  the  same 
time. 

Film  preparations  are  also  made. 

(4)  Incubate  the  plates  till  the  following  morning. 

(5)  Examine  the  films  and  the  plates  for  the  predominant 
organisms. 

Pick  off  with  the  platinum  wire  sample  colonies  from  the 
plate,  and  subculture  them. 

(6)  Subculture  from  the  secondary  cultures  into  the 
appropriate  media  organisms  likely  to  be  of  pathological 
importance.  Ignore  colonies  of  sarcinae,  spore-bearing  bacilli, 
and  in  most  cases  staphylococci. 

If  delicately-growing  organisms  of  the  influenza  bacillus 
type  are  being  sought  for,  substitute  nasgar  for  agar  plates 
in  stage  3. 

Among  the  organisms  to  be  looked  for  in  the  sputum  are 
the  following : — 

The  tubercle  bacillus. — The  methods  of  examining  the 
sputum  for  tubercle  bacilli  have  been  already  described 
(page  155).  If  there  is  any  urgency  films  may  be  made  from  the 
sputum  direct,  and  stained  by  the  Ziehl-Neelsen  process.  In  the 
majority  of  cases  it  is  preferable  to  carbolise  the  sputum  and 
let  it  stand  till  the  next  day.  In  those  cases  in  which  tubercu- 
losis is  strongly  suspected  and  there  is  an  appreciable  quantity 
of  sputum,  yet  no  tubercle  bacilli  have  been  found  by  the 
carbolic  acid  process,  a  further  sample  of  sputum  may  be 
treated  by  the  "  antiformin  "  method.  It  is,  however,  excep- 
tional to  detect  bacilli  in  a  sample  of  sputum  after  treating 
with  antiformin  if  they  have  not  previously  been  found  after 
carbolising.  It  occasionally  happens,  on  the  contrary,  that 
scanty  bacilli  are  found  in  the  carbolised,  and  not  in  the 
antiformin,  films. 

In  all  doubtful  cases  at  least  15  minutes  should  be  given  to 

p.  21 


370  CLINICAL   PATHOLOGY. 

each  slide  before  abandoning  the  search,  and  it  may  be 
necessary  to  examine  the  morning  sputum  on  two  or  three 
occasions. 

If  the  patient  with  suspected  pulmonary  tuberculosis  is  a 
child  who  swallows  the  sputum  it  is  worth  while  to  examine 
the  faeces  for  the  bacilli  by  the  antiformin  method. 

The  pneumococcus.— The  diagnosis  of  lobar  pneumonia 
by  the  demonstration  of  the  causative  organism  in  the 
sputum  is  rarely  called  for,  and  fortunately,  since  the  bacterio- 
logical diagnosis  is  unsatisfactory.  In  films  made  from  the 
sputum  a  variety  of  organisms  are  often  present,  and  the 
pneumococcus  can  rarely  be  identified  with  any  certainty.  In 
cultures  streptococci  are  often  found  in  addition  to  the  pneu- 
mococci,  and  the  colonies  of  these  organisms  are  so  similar 
that  it  is  difficult  to  separate  them.  Further,  pneumococci 
may  be  obtained  from  the  sputum  in  other  conditions  than 
lobar  pneumonia. 

There  is  little  question  that  the  great  majority  of  cases  of 
lobar  pneumonia  are  produced  by  the  pneumococcus,  as  can  be 
shown  by  lung  puncture  and  by  the  examination  of  empyema 
fluids.  A  minority  of  cases,  however,  are  due  to  streptococci 
and  other  organisms.  Streptococcal  pneumonia  is  commoner 
in  children  than  in  adults,  and  more  often  has  a  broncho- 
pneumonic  than  a  lobar  distribution. 

The  recognition  of  the  pneumococcus  requires  cultural 
investigation,  and  should  not  rest  with  the  appearance  of  the 
organism  in  film  preparations. 

Friedlander's  pneumo-bacillus. — This  organism  is  pre- 
sent in  the  sputum  in  some  cases  of  pneumonia,  as  well  as  in 
other  conditions.  It  may  be  found  also  in  the  mouth  and  in 
the  nasal  cavity. 

The  bacilli  are  Gram-negative  capsulated  organisms,  which 
usually  appear  as  short  rods  with  rounded  ends.  They  occur 
in  the  films  in  small  groups  of  2,  3,  and  4. 

The  pneumo-bacillus  is  best  isolated  from  the  sputum  by 
plating  direct  on  two  gelatine  plates.  The  colonies  appear  as 
white,  heaped-up  points  on  the  plates,  and  there  is  no  liquefac- 
tion of  the  gelatin  In  gelatin  stab  cultures  growth  occurs 
along  the  track  of  the  inoculating  wire,  and  profusely  in  a 
round,  heaped-up  growth  at  the  surface  of  the  stab.  The 
growth  in  gelatin  stab  cultures  is  described  as  "  nail  shaped." 


THE   NOSE— THE    SPUTUM.  371 

Litmus  milk  is  acidified  and  clotted.  A  copious  and  viscid 
growth  is  obtained  on  an  agar  slope.  Both  acid  and  gas  are 
produced  in  the  litmus  carbo-hydrate  media. 

The  influenza  bacillus. — The  bacilli  in  the  earlier  stages 
of  the  disease  may  be  found  in  very  large  numbers  in  the 
sputum.  They  may  occur  in  considerable  sized  clumps  out- 
side the  cells,  and  a  minority  of  them  are  intracellular.  The 
minute  "  dew  drop  "  colonies  may  be  obtained  in  plate  cultures 
on  glycerine  agar,  or  preferably  on  serum  agar  or  nasgar. 
The  colonies  grow  somewhat  slowly,  and  the  organisms,  unless 
particularly  abundant,  are  apt  to  be  overgrown  by  other 
bacteria. 

Gram-stained  film  preparations  of  the  sputum  counterstained 
with  carbol  fuchsin  are  useful  as  a  means  of  diagnosis.  The 
minute  red  bacilli  are  sufficiently  characteristic. 

Streptococci. — These  organisms  are  very  constantly  met 
with  in  the  sputum,  but  are  usually  not  of  the  pyogenes  variety. 
Perhaps  the  commonest  bacterium  met  with  in  all  samples  of 
sputum  is  a  short-chained  streptococcus  of  the  brevis  type, 
which  acidifies  and  clots  milk  and  which  resembles  the  pneu- 
mococcus  in  its  cultural  characters  more  closely  than  the 
Streptococcus  pyogenes  of  acute  inflammation.  The  organism 
differs  markedly  from  the  pneumococcus  in  its  low  pathogenicity 
to  mice  and  rabbits.  The  Streptoccus  brevis  probably  plays 
a  part  in  a  variety  of  pulmonary  infections,  but  mainly  as  a 
secondary  invader  of  the  lung.  It  is  an  organism  often  pre- 
dominant in  the  contents  of  a  bronchiectatic  cavity. 

Staphylococci. — These  organisms  are  frequently  found 
in  the  sputum,  and  may  be  responsible  for  a  percentage  of 
bronchitis  cases.  S.  aureus  and  S.  citreus  are  much  less 
commonly  met  with  than  the  white  staphylococcus,  which  is 
found  in  practically  all  sputa  and  has  no  diagnostic  signifi- 
cance. 

Diphtheria  bacillus. — This  bacillus  is  rarely  found  in  the 
sputum,  but  in  cases  of  laryngeal  diphtheria  with  a  membrane 
spreading  downwards  portions  of  the  membrane  may  be  occa- 
sionally coughed  up.  Membrane  is  commonly  expelled  from  a 
tracheotomy  wound,  and  there  is  little  difficulty  in  cultivating 
the  bacillus  from  it. 

Actinomyces. — Actinomycosis  of  the  lung  is  an  uncommon 
condition,  but  the  organisms  may  obtain  primary  lodgment 

24—2 


372  CLINICAL   PATHOLOGY. 

in  the  lung  or  may  be  carried  there  from  a  distant  focus.  The 
characteristic  granules  can  be  found  in  the  sputum  or  in  the 
pus  from  an  empyema,  and  should  be  examined  in  the  ordinary 
way.  The  beaded  Gram-positive  streptothrix  is  sufficiently 
characteristic,  but  "  clubs  "  are  practically  never  met  with. 
Other  streptothrices  are  occasionally  found,  and  some  may  be 
both  Gram-positive  and  acid-fast,  thus  resembling  the  long 
and  beaded  forms  of  the  tubercle  bacillus.  The  diagnosis  of 
a  streptothrix  infection  from  the  sputum  must  be  made  with 
care,  since  streptothrix-like  organisms  may  be  present  in  the 
mouth  as  a  contamination  from  the  food,  and  it  is  advisable 
that  the  mouth  should  be  thoroughly  cleansed  before  the 
sample  of  sputum  is  obtained.  The  actinomyces  organism 
occurs  in  tufts  (see  Plate  IX.)  of  beaded  filaments,  and  should 
not  be  confounded  with  the  long,  thin  beaded  bacilli  often 
present  in  sputum,  nor  with  the  fine  fibrinous  filaments  seen 
in  all  film  preparations  from  sputum  or  muco-pus. 

The  plague  bacillus. — In  the  pneumonic  forms  of  plague 
the  causative  organism  is  to  be  met  with  in  large  numbers  in 
the  sputum.  The  bacilli  are  present  also  in  the  septicemic 
form  of  plague,  and  in  the  bubonic  form,  but  only  in  the 
latter  condition  when  there  is  a  pyaemia  with  metastatic 
abscesses  in  the  lung.  The  diagnosis  in  a  plague  district  may 
reasonably  be  made  from  the  appearance  of  the  bacilli  in  film 
preparations.  The  bacilli  occur  in  the  sputum  in  pairs,  clumps, 
and  short  chains.  The  bipolar  staining  is  best  seen  if  the 
films  are  fixed  in  absolute  alcohol  before  staining.  Cultures 
of  the  bacillus  should  be  obtained  if  possible,  but  the  organism 
is  often  oul grown  by  the  other  bacteria  of  the  sputum. 

Lung*  puncture. — Puncture  of  the  lung  through  the  chest 
wall  as  a  means  of  diagnosis  has  already  been  referred  to,  but 
since  it  is  a  proceeding  not  devoid  of  risk  is  rarely  justifiable. 
The  operation  is  not  infrequently  performed  accidentally  in  the 
expectation  of  finding  fluid  in  the  pleural  cavity,  and  the  small 
quantity  of  blood-stained  fluid  removed  from  the  lung  is  often 
sufficient  for  film  preparations  and  for  cultural  processes.  The 
bacteriological  examination  of  fluid  removed  in  this  way  is 
more  satisfactory  than  similar  investigations  of  the  sputum. 

Pleural  fluids. — The  nature  and  mode  of  examination  of 
these  fluids  has  been  described  in  the  section  on  "  Puncture 
Fluids." 


SECTION   VIII. 

HISTOLOGY. 

CHAPTEE  XXVI. 

The  Examination  of  Sections — The  Inflammations — The  Degenerations. 

CHAPTEE  XXVII. 

Neoplasms — Simple  Tumours. 

CHAPTEE   XXVIII. 

Carcinomata — Sarcomata — Other  Tumours — Cysts. 

CHAPTEE  XXIX. 

Histological  Methods. 


CHAPTER  XXVI. 

the     examination     of      sections the     inflammations the 

degeneeations. 

The  Examination  of  Sections. 

The  histological  examination  of  tissues  removed  during 
life  is  among  the  most  important  of  the  methods  of 
clinical  pathology,  and  from  the  point  of  view  of  diag- 
nosis probably  the  most  difficult.  There  is  no  other 
method  of  diagnosis  which  requires  more  experience  for  the 
interpretation  of  what  one  sees,  and  the  necessary  experience 
cannot  be  gained  by  reading,  but  comes  from  repeated 
examination  of  many  types  of  the  same  pathological  change. 

An  acquaintance  with  the  normal  histology  of  the  tissues  is 
essential,  and  the  student  is  advised  not  only  to  examine, 
whenever  possible,  type  specimens  of  tumours  and  granulo- 
mata,  but  also  to  renew  his  acquaintance  with  sections  of  the 
normal  human  tissues. 

Methods  of  examining  sections. — It  is  a  common 
elementary  crime  of  clinical  medicine  for  the  beginner,  on 
being  asked  to  examine  a  patient,  to  forget  that  he  is  provided 
with  eyes  and  hands  and  to  immediately  clap  a  stethoscope  on 
the  chest.  A  very  similar  error  is  made  in  histological 
diagnosis.  The  impulse  of  the  novice  drives  him  to  place  his 
section  on  the  largest  available  microscope  and  examine  it  at 
once  under  the  highest  possible  power.  The  impulse  is  possibly 
fostered  by  a  deplorable  "  examination "  process  vulgarly 
known  as  "  spotting  "  sections.  The  most  convenient  micro- 
scope for  the  majority  of  histological  work  is  a  small  one  with- 
out a  mechanical  stage.  The  most  suitable  powers  are  a  No.  2 
eye-piece  with  a  §-inch  and  J-inch  objective.  An  ordinary 
hand  magnifying  glass  is  often  of  considerable  assistance. 
Higher  magnifying  powers  are  occasionally,  though  rarely, 
required. 

The  sections  should  first  be  examined  with  the  naked  eye, 
and  points  of  considerable  importance  are  frequently  to    be 


THE   EXAMINATION   OF    SECTIONS,   ETC.      375 

made  out.  The  naked-eye  inspection  can  be  amplified  by- 
holding  the  slide  against  the  light  and  examining  it  further  with 
the  hand  glass.  The  distinction  between  the  normal  tissue 
and  the  abnormal  is  often  obvious,  and  the  general  aspect  of 
the  section  is  of  great  value  when  considering  the  microscopic 
appearance  of  a  series  of  "  fields."  The  entire  area  of  the 
section  is  then  carefully  gone  over  with  the  §-inch  objective. 
Under  this  power  the  relation  of  the  normal  to  the  abnormal 
tissues  is  definitely  made  out,  the  grosser  structures  are 
recognised,  and  to  a  less  extent  the  relation  of  the  cellular 
elements  to  the  connective  tissue  is  observed. 

The  ^-inch  objective  is  used  last  and  most  sparingly.  The 
portions  of  the  section  to  be  examined  more  particularly  will 
have  been  indicated  by  the  previous  inspection.  The  points 
to  be  considered  are  the  nature  of  the  cells,  their  size,  shape,  and 
staining  reaction,  and  the  character  of  their  nuclei.  The  rela- 
tion of  the  cells  to  each  other  and  to  the  connective  tissue 
network  is  further  investigated.  The  observer  who  pays  too 
much  attention  to  the  higher  magnification  and  too  little  to  the 
general  examination  under  the  lower  powers  is  in  danger  of 
"  not  seeing  the  wood  for  the  trees."  Further,  in  considering  the 
section,  the  observer  must  not  lose  sight  of  the  patient.  All 
the  available  clinical  information  concerning  the  case  must  be 
made  use  of,  and  the  naked-eye  appearance  of  the  tissues 
removed  must  be  considered. 

The  following  brief  description  of  the  various  processes  of 
morbid  histology  is  not  intended  in  any  sense  to  give  a 
detailed  account  of  the  various  changes  which  may  occur.  It 
is  only  possible  to  indicate  the  variety  of  processes  which  may 
be  met  with,  and  the  more  important  points  to  be  considered 
in  the  microscopical  diagnosis. 

The    Inflammations. 

The  inflammations  may  be  divided  into  the  acute  and  the 
chronic  granulomata.  The  acute  granuloraata  are  considered 
under  the  heading  of  "  acute  inflammation,"  and  the  chronic 
granulomata  include  the  lesions  of  tuberculosis,  syphilis, 
leprosy,  actinomycosis,  and  glanders.  Lymphadenoma,  or 
Hodgkin's  disease,  is  also  conveniently  considered  here, 
since  our  ignorance  of  the  essential  nature  of  the  condition 
prevents  a  more  definite    classification.     A  short  account  is 


376  CLINICAL   PATHOLOGY. 

also  given  of  molluscum  contagiosum,  another  disease  of 
unknown  aetiology. 

Acute  inflammation. — By  acute  inflammation  is  meant 
the  usual  series  of  changes  found  in  a  tissue  as  the  result 
of  irritation  by  the  toxins  of  pyogenic  organisms,  or  less 
commonly  by  mechanical  irritants.  The  word  "  acute  "  is  used 
in  the  pathological  rather  than  in  the  clinical  sense  to  indicate 
a  particular  type  of  change.  The  processes  involved  may 
continue  for  a  sufficient  length  of  time  to  justify  the  clinical 
description  of  "  chronic."  The  term  "  granuloma  "  is  commonly 
used  to  describe  inflammation  accompanied  by  the  formation 
of  granulation  tissue,  and  due  to  one  of  the  specific  diseases 
referred  to  here  under  the  heading  of  "  chronic  granulomata." 
Granulation  tissue  is,  however,  a  frequent  product  of  the  acute 
variety  of  inflammation. 

The  typical  changes  of  acute  inflammation  are  most 
commonly  produced  by  the  ordinary  pyogenic  organisms,  such 
as  staphylococci  and  streptococci. 

The  tissue  most  commonly  removed  for  diagnostic  purposes 
is  granulation  tissue  from  the  more  long  standing  varieties  of 
acute  inflammation.  The  histological  diagnosis  usually  lies 
between  the  acute  and  chronic  granulomata  and  some  form  of 
new  growth,  particularly  sarcoma. 

The  changes  to  be  met  with  involve  the  special  tissue 
elements  affected,  the  blood-vessels,  and  the  cellular  elements. 

The  tissue  changes  are  mainly  degenerative,  and  range 
from  cloudy  swelling  to  actual  necrosis.  Glandular  cells,  for 
example,  stain  poorly,  both  as  regards  their  cytoplasm  and 
their  nuclei,  and  often  show  fatty  change.  Muscle  cells  stain 
homogeneously,  and  lose  their  striation.  The  changes  of 
repair  include  the  regeneration  and  multiplication  of  the  cells 
of  the  less  specialised  tissues,  and  the  linking  of  breaches  by 
fibrous  tissue.  The  stage  of  fibrosis,  in  which  the  cellular 
infiltration  has  largely  disappeared,  is  less  commonly  seen  in 
the  clinical  laboratory  than  the  cellular  granulomatous  stage 
of  still  active  inflammation. 

The  vascular  changes  are  often  far  from  obvious.  The 
earlier  changes  of  dilatation,  alteration  in  the  rate  of  flow,  and 
diapedesis  of  cells  are  necessarily  for  the  most  part  lost  at  the 
moment  of  removing  the  tissue  from  the  body ;  thus  a  section 
taken    through    the    red    and    obviously    inflamed    skin  in 


THE   EXAMINATION   OP   SECTIONS,   ETC.      377 

erysipelas  may  show  practically  no  change  in  the  actual 
vessels  themselves.  In  the  more  chronic  processes  the  tissues 
often  show  marked  vascularity  owing  to  the  abundant  for- 
mation of  new,  thin-walled  vessels,  which  at  first  appear  as 
mere  clefts  in  the  tissue  meshwork  lined  with  a  single  layer,  or 
with  a  few  layers  only,  of  endothelial  cells  and  filled  with  red 
corpuscles.  Well-formed  vessels  are  usually  present  in 
addition. 

The  cellular  changes  are  the  most  obvious  and  important 
in  the  histological  examination.  The  cells  are  of  several 
varieties,  and  are  derived  partly  from  the  interior  of  the  blood- 
vessels in  the  inflamed  area,  by  active  diapedesis  of  the 
leucocytes  and  passive  exudation  of  the  red  cells,  and  partly 
from  the  local  tissue  cells.  The  cells  may  be  scattered  at 
random  through  the  loose  connective  tissue  of  a  granulation, 
or  collected  into  circular  areas,  forming  the  microscopical 
abscesses  of  a  local  pyaemic  condition,  as  may  be  well  seen  in 
some  forms  of  the  so-called  surgical  kidney. 

The  cells  other  than  red  corpuscles  consist  mainly  of  the 
ordinary  polynuclear  neutrophil  cells  of  the  blood.  The 
more  acute  the  inflammatory  process,  the  more  abundant  and 
predominant  are  the  polynuclears.  These  cells,  in  common 
with  the  lymphoid  cells  shortly  to  be  described,  are  referred 
to  in  many  descriptions  as  the  "  small  round  cells  "  of  in- 
flammation. It  is  preferable  to  give  them  the  name  by  which 
they  are  known  in  their  natural  surroundings,  and  it  is 
necessary  to  differentiate  them  from  other  varieties  of  "  round 
cells."  In  a  paraffin  section  stained  with  hematoxylin  and 
eosin  the  polynuclear  neutrophils  are  readily  recognised 
under  the  J-inch  objective.  They  appear  as  small 
round  cells,  with  an  eosinophilic  cytoplasm  and  a  bilobed,  or 
less  commonly  trilobed,  nucleus. 

Cells  derived  partly  from  the  blood  and  partly  from  the 
tissues  are  "  lymphoid  "  and  "  endothelioid  "  cells. 

The  lymphoid  cells  are  possibly  of  two  varieties,  derived  in 
part  from  the  lymphocytes  of  the  blood  and  in  part  from 
similar  cells  of  the  tissues.  They  appear  as  small  round  cells, 
with  very  little  cytoplasm  and  a  relatively  large  deeply-stain- 
ing round  nucleus.  They  are  found  only  in  very  small 
numbers  in  the  early  stages  of  acute  inflammation. 

Endothelioid    cells,    or    as   they   are   sometimes   called 


378  CLINICAL   PATHOLOGY. 

epithelioid  cells,  are  probably  of  numerous  varieties.  The 
majority  of  them  are  wandering  phagocytic  cells.  They  are 
derived  in  part  from  the  large  hyalines  of  the  blood,  in  part 
from  vascular  and  other  endothelial  membranes,  and  in  part 
from  the  cells  of  the  local  tissue.  They  have  probably  a 
similar  origin  to  the  elongated  tissue  fibroblast.  They 
aj)pear  as  large  cells,  often  of  a  more  or  less  angular  shape. 
The  cytoplasm  is  clear,  relatively  abundant,  and  takes  the 
eosin  dye  faintly.  The  nuclei  are  comparatively  small,  faintly 
staining,  and  often  show  nucleoli ;  they  may  be  round  or 
slightly  indented. 

An  important  cell  of  inflammation,  and  one  which  is 
probably  derived  from  the  tissues,  is  the  plasma  cell.  The 
origin  and  functions  of  the  plasma  cell  are  still  in  dispute  and 
do  not  concern  us  here.  It  is  probably  mainly  derived 
from  the  primitive  perivascular  lymphocytic  cell  of  the 
neighbourhood.  Special  stains  are  in  use  for  the  demon- 
stration of  plasma  cells,  but  they  are  readily  distinguished  in 
the  ordinary  hematoxylin  and  eosin  preparations.  The  cells 
are  intermediate  in  size,  between  the  lymphoid  and  endo- 
thelioid  cells.  Their  shape  is  more  or  less  that  of  an  egg, 
and  their  cytoplasm  is  distinctly  eosinophilic,  staining  a  bright 
pink.  Their  nuclei  are  characteristically  eccentric,  and  appear 
as  if  they  were  in  the  process  of  being  squeezed  out  of  the  cell. 
Plasma  cells  are  nearly  always  present  in  acute  inflammatory 
processes,  and  are  fairly  abundant  in  those  of  some  standing. 

In  addition  to  the  tissue  changes  of  inflammation  the  exciting 
cause  must  often  be  looked  for.  The  examination  for 
organisms  can  be  conducted  by  Gram's  method  of  staining 
applied  to  tissues,  or  by  a  simple  .process  with  carbol-thionin. 
Both  these  methods  will  be  described  subsequently.  Organisms 
may  be  present  in  large  clumps  visible  under  the  ^-inch, 
or  even  the  §-inch  objective,  but  it  is  necessary  to  use 
the  oil  immersion  lens  in  order  to  determine  their  morphology. 

In  sections  through  an  anthrax  pustule,  for  example,  large 
masses  of  big  Gram-positive  bacilli  are  usually  conspicuous. 
The  exact  nature  of  organisms  can  only  exceptionally,  as  in 
the  case  of  the  tubercle  bacillus,  be  identified  in  sections ;  nor 
does  the  failure  to  demonstrate  bacteria  in  sections  invalidate 
other  evidence  of  a  bacterial  infection. 

In  examining  a  section  of  granulation  tissue  from  an  acute 


THE   EXAMINATION   OF    SECTIONS,   ETC.      379 

inflammatory   focus   the   points   to   be   looked   for  are    con- 
sequently the  following :  — 

A  loose  connective  tissue  basis  enclosing  an  abundance  of 
newly-formed  blood-vessels.  A  scattered  and  irregular  cellular 
infiltration,  composed  mainly  of  polynuclear  neutrophils,  with 
occasional  plasma  cells,  few  lymphoid  cells,  and  an  increasing 
number  of  endothelioid  cells  and  fibroblasts,  according  to  the 
duration  of  infection.  Free  red  cells  are  usually  present  in  the 
connective  tissue  spaces.  The  adjacent  tissues  of  the  part 
show  degenerative  changes  in  greater  or  less  degree.     Micro- 


0. 

*> 

*■■ 

. 

%g 

0 

jf$ 

n 

**  * 

9jf, 

tic 

Fig.  28. — Acute  Infective  Granuloma.     Showing  Polynuclear  and 
Plasma  Cells.     Drawn  under  £-inch  Objective. 

organisms  may  be  present  in  considerable  numbers.  The 
most  characteristic  changes  of  all  are  the  presence  of  bacteria, 
and  the  preponderance  of  polynuclear  neutrophils. 

Tuberculosis. — Tuberculosis  produces  the  commonest  and 
most  widely  spread  lesions  of  the  chronic  infective  granu- 
lomata.  Almost  any  part  of  the  body  may  be  affected,  but  the 
changes  are  practically  identical  in  all  tissues. 

The  characteristic  histological  process  resulting  from  a 
tuberculous  infection  is  the  giant  cell  system  or  miliary 
tubercle.  Other  changes  are  either  antecedent  to  this,  or 
result  from  the  spread  or  arrest  of  the  infection.     Thus  in  the 


380  CLINICAL   PATHOLOGY. 

very  earliest  stages  there  may  be  a  proliferation  of  endothelioid 
cells,  but  no  giant  cell  system,  and  in  the  later  stages  the 
giant  cell  systems  may  increase  in  size  by  enlargements  of  their 
caseous  centres  and  coalesce  with  adjoining  systems  to  form 
tuberculous  foci  of  irregular  shape  and  size.  Caseation  and 
fibrosis  commonly  proceed  together,  and  both  are  features  of  a 
section  through  an  advanced  tuberculous  lesion. 

The  miliary  tubercle  is  in  a  section  just  visible  to  the  naked 
eye,  and  its  zones  can  be  made  out  under  the  magnification  of 
a  hand  glass.  The  gross  appearance  of  a  section  containing 
two  or  three  such  tubercles  is  very  characteristic. 

The  giant  cell  system  has  the  following  structure  : — In  the 
centre  is  a  giant  cell.  Surrounding  the  giant  cell  is  a  zone  of 
endothelioid  cells.  Surrounding  the  endothelioid  cells  is  a 
zone  of  lymphoid  cells.  Blood-vessels  are  absent.  As  the 
tubercle  grows  larger  its  centre  caseates.  Two,  three  or 
more  giant  cells  lie  in  the  periphery  of  the  caseous  centre,  and 
outside  these  again  are  the  endothelioid  and  lymphoid  cells. 

The  caseous  centre  of  the  tubercle  stains  a  bright  pink  with 
the  eosin  dye. 

The  giant  cell  of  a  tuberculous  lesion  is  very  characteristic. 
It  has  an  irregular  and  often  angular  shape,  but  the  larger  cells 
in  particular  are  often  rounded.  Its  centre  is  caseous  and 
stains  a  pinkish  colour  with  eosin.  It  is  multinucleated  and 
may  contain  10  or  more  nuclei.  The  nuclei  are  usually  confined 
to  and  arranged  round  the  periphery  of  the  cell.  The  larger  the 
giant  cell,  as  a  rule,  the  more  caseous  its  centre,  and  the  more 
regular  the  arrangement  of  nuclei  round  its  periphery. 
Smaller  and  less  characteristic  giant  cells  are  often  present  in 
addition,  and  these  may  contain  fewer  nuclei,  some  of  which 
have  a  nearly  central  position.  The  large  giant  cell  is,  with 
the  exception  of  the  demonstration  of  the  tubercle  bacillus, 
the  most  diagnostic  histological  feature  of  a  tuberculous  lesion. 
It  is  easily  visible  under  the  low  power  of  the  microscope,  and 
may  attain  such  a  size  as  to  occupy  the  greater  part  of  the 
field  of  a  ^-inch  objective. 

Giant  cells  are  present  in  a  variety  of  other  conditions. 
They  occur  in  all  the  chronic  infective  granulomata,  including 
syphilis,  but  are  as  a  rule  very  scarce,  comparatively  small,  and 
without  the  typical  nuclear  arrangement  found  in  tuberculosis. 
In  Hodgkin's  disease  giant  cells,  of  a  type  to  be  subsequently 


THE   EXAMINATION   OF    SECTIONS,   ETC.      381 

described,  are  numerous,  but  the  nuclei  are  central  and  the 
cells  do  not  at  all  resemble  those  of  tuberculosis.  Foreign 
bodies,  such  as  ligatures,  commonly  have  formed  round  them 
regular  giant  cell  systems  which  very  closely  resemble  miliary 


..•■;■    ■■■'   %y,,,;  ■','$//:<.  '  :      «  ,       ' :   .•  ..    ■"  .' 


'    Sj>»- 


W:--M/8i 


'/■&;:• 


■>''  '•'  ■■•.'•'''."•'•f:; !'"."'.  ■'■','.■■  v- .'" !', 


Fig.  29. — Tuberculosis  of  Kidney.     Drawn  under  §-inch  Objective. 


Fig.  30. — High  Power  Drawing  of  a  giant  Cell 
in  Fig.  29. 

tubercles.  The  giant  cells  as  a  rule  have  nuclei  placed  cen- 
trally as  well  as  peripherally,  and  a  careful  examination  often 
discovers  the  foreign  body,  which  may  be  engulfed  within  one 
of  the  giant  cells.     Portions  of  ligature  in  a  section  show  as 


382  CLINICAL   PATHOLOGY. 

glistening  threads,  often  faintly  stained  with  the  haeniatoxylin 
dye.  Similar  giant  cells  may  also  be  found  in  lesions  resulting 
from  some  chronic  irritant,  as  at  a  point  of  pressure,  the  site  of 
a  chronic  septic  discharge,  or  the  edge  of  a  new  growth.  The 
giant  cells  of  myeloid  sarcoma  are  large  cells  with  nuclei 
evenly  scattered  throughout  their  substance. 

In  sections  of  tuberculous  tissues  removed  during  life  regular 
giant  cell  systems  are  often  absent.  The  tissues  show  areas 
of  fibrosis,  and  irregular  caseous  areas  merging  into  zones  of 
cellular  infiltration.  Giant  cells  are  to  be  looked  for  in  the 
margins  of  the  caseous  areas.  The  cellular  infiltration  is  com- 
posed mainly  of  lymphoid  and  endothelioid  cells.  Polynuclears 
and  plasma  cells  are  usually  scanty  or  absent,  but  a  few  are 
often  found  at  the  periphery  of  a  tuberculous  area,  and  if  a 
secondary  infection  has  occurred  they  may  be  numerous.  The 
histological  appearance  of  a  tuberculous  lesion  is  in  the  majority 
of  cases  sufficient  for  diagnostic  purposes.  In  some  cases, 
however,  it  is  desirable  to  examine  for  the  bacillus.  The  method 
of  staining  the  bacilli  in  section  is  described  later.  The 
number  of  bacilli  present  in  the  majority  of  tuberculous  sections 
is  extremely  small,  and  to  find  a  single  bacillus  on  one  slide  is 
about  as  much  as  can  be  hoped  for.  Failure  to  find  the  bacilli 
is  consequently  little  evidence  against  a  diagnosis  of  tuber- 
culosis. 

The  points  in  the  histological  diagnosis  of  tuberculosis 
therefore  include  the  presence  of  giant  cell  systems  in  an  earl}7 
lesion,  with  later  areas  of  caseation  and  fibrosis,  and  a  cellular 
infiltration  consisting  mainly  of  lymphoid  and  epithelioid  cells. 

Syphilis. — The  changes  produced  in  the  tissues  by  syphilis 
are  varied,  and  include  nothing  so  characteristic  to  the 
histologist  as  the  giant  cell  system  of  tuberculosis.  The  certain 
diagnosis  of  a  syphilitic  lesion  on  histological  grounds  is  often 
impossible,  and  one  may  only  be  able  to  state  that  the  lesion  is 
due  to  one  of  the  chronic  infective  granulomata. 

Syphilitic  tissues  are  not  commonly  removed  during  life,  and 
a  very  brief  account  of  the  changes  to  be  met  with  is  given 
here. 

The  tissue  changes  of  syphilis  consist,  as  in  tuberculosis,  of 
caseation  and  fibrosis.  A  diffuse  and  irregular  fibrosis  is  a 
characteristic  feature  of  many  syphilitic  tissues. 

The  vascular  changes  in  sj'philitic  lesions  are  nearly  always 


THE   EXAMINATION   OF    SECTIONS,   ETC.      383 

very  noticeable.  The  smaller  arterioles  have  their  lumina 
markedly  narrowed  by  proliferation  of  the  cells  of  the  intima, 
and  there  is  often  fibrosis  and  thickening  of  the  vessel  walls, 
with  an  infiltration  of  lymphoid  cells  into  the  media,  as  well 
as  into  the  adjacent  tissues. 

The  cellular  exudation  into  the  tissues  is,  except  in  the 
advanced  fibrotic  cases,  very  considerable.  The  cells  consist 
of  lymphoid,  endothelioid,  and  plasma  cells.  The  plasma  cells 
are  often  very  numerous,  in  contradistinction  to  what  is  com- 
monly found  in  tuberculous  lesions.  Giant  cells  are  usually 
scanty  or  absent,  and  it  is  most  unusual  to  find  in  syphilitic 
lesions  large  giant  cells  with  peripheral  nuclei  such  as  are 
seen  in  tuberculomata.  The  main  changes  in  a  syphilitic 
lesion  are  the  same  in  all  stages  of  the  disease,  but  differ  some- 
what in  detail. 

A  section  through  the  primary  chancre  shows  a  formation 
of  new  capillaries  running  at  right  angles  to  the  ulcerated 
surface,  a  proliferation  of  the  intima  of  the  arterioles,  and 
between  the  capillaries  young  fibrous  tissue  and  fibroblasts, 
together  with  a  large  number  of  lymphoid  cells  and  plasma 
cells,  which  spread  also  into  the  adjacent  tissues. 

The  mucous  tubercles  and  condylomata  of  secondary 
syphilis  are  produced  by  the  accumulation  of  cells  and  exuded 
fluid,  together  with  a  proliferation  of  the  overlying  epithelial 
cells. 

Tertiary  syphilis  is  characterised  by  the  formation  of 
gummata  consisting  of  circular  caseous  areas  surrounded 
by  fibrous  tissue  and  an  area  of  cellular  infiltration. 

The  points  to  be  looked  for  in  the  sections  of  a  syphilitic 
lesion  are  caseation,  fibrosis,  arterial  changes,  and  particularly 
a  proliferation  of  the  intima  of  the  arterioles,  and  a  cellular 
infiltration  by  lymphoid  and  plasma  cells. 

The  Spirochceta  pallida  can  be  demonstrated  in  the  primary 
and  secondary,  but  not  in  the  tertiary,  lesions. 

Leprosy. — The  cases  of  leprosy  met  with  in  this  country 
are  few,  and  all  are  imported.  The  lesions  examined  during 
life  usually  consist  of  nodules  from  the  skin.  The  cellular 
infiltration  is  considerable,  and  the  predominant  cell  is  of  the 
endothelioid  variety.  Caseation  and  fibrosis  occur,  and  giant 
cells  may  be  found  in  considerable  numbers.  The  diagnosis 
of  the  condition  on  histological  grounds  is  as  a  rule  readily 


384  CLINICAL   PATHOLOGY. 

made  by  the  demonstration  of  acid-fast  bacilli  in  the  sections. 
The  bacilli  are  stained  by  the  same  method  as  that  given  for 
tubercle  bacilli  hi  section  (page  432),  but  12  per  cent,  acid  must 
be  used  for  decolorising  in  iilace  of  25  per  cent.  The  bacilli 
are  usually  numerous  in  sections,  and  are  often  packed  within 
large  epithelioid  cells. 

Actinomycosis. — Actinomycotic  nodules  are  rarely  met 
with  in  human  pathology,  but  are  exceptionally  found  in  the 
skin,  and  not  very  infrequently  the  granuloraata  may  be  found 
in  the  appendix  or  in  scrapings  from  an  abscess  of  the  liver. 
The  diagnosis  of  the  condition  rests  upon  the  discovery  of  the 
organism,  and  the  threads  are  most  readily  found  in  the  pus, 
but  may  appear  in  large  numbers  and  in  clumps  of  consider- 
able size  in  the  sections.  The  sections  should  be  stained  by 
Gram's  method  (page  432).  The  histology  of  the  lesion  is  not 
particularly  characteristic  in  the  absence  of  proof  of  the 
causative  organism.  There  is  a  considerable  infiltration  of 
the  tissues  forming  the  nodule  by  lymphoid  cells,  which  are 
subsequently  replaced  by  polynuclear  neutrophils.  The  central 
portion  breaks  down  to  form  pus.  Giant  cells  and  fibroblasts 
are  only  exceptionally  present,  but  the  inflammatory  exudate 
may  become  encapsuled  by  fibrous  tissue  and  the  central  area 
calcined. 

Glanders- — Glanders  is  properly  a  disease  of  equines,  and 
is  rarely  conveyed  from  them  to  man.  The  glanders  nodule 
has  a  central  area  of  necrosed  polynuclear  cells,  a  middle  zone 
of  endothelioid  cells  with  occasional  giant  cells,  and  an  outer 
ring  of  young  fibrous  tissue.  The  nodules  readily  break  down 
with  the  formation  of  ordinary  pus.  The  diagnosis  of  the 
condition  rests  with  the  isolation  of  the  specific  bacillus. 

Hodgkin's  disease  (Lymphadenoma).— The  aetiology  of 
Hodgkin's  disease  is  unknown.  The  condition  has  been 
classified  among  the  blood  diseases  from  its  supposed  relation- 
ship to  lymphatic  leukaemia,  but  the  two  conditions  have 
practically  nothing  in  common.  It  has  been  considered  a 
form  of  new  growth,  allied  to  the  sarcomata,  owing  to  its 
usually  rapid  dissemination  and  fatal  termination.  The 
modern  consensus  of  opinion  probably  is  that  Hodgkin's 
disease  will  prove  to  be  a  parasitic  disease,  and,  in  accordance 
with  that  view,  it  is  conveniently  considered  here. 

The  disease  affects  the  lymphatic  tissues,  at  first  locally,  as  in 


THE    EXAMINATION  OF    SECTIONS,   ETC.      385 

the  glands  of  the  neck,  and  later,  as  a  rule,  more  or  less  generally 
throughout  the  body. 

The  diagnosis  of  the  condition  can  only  be  made  with  cer- 
tainty on  histological  grounds,  and  for  this  reason  one  of  the 
discrete  superficial  glands  may  be  removed  under  a  local 
anaesthetic.  The  enlarged  glands,  if  localised,  are  removed 
by  the  surgeon  as  a  mode  of  treatment. 

The  histological  picture  is  a  characteristic  one. 

Under  the  low  power  of  the  microscope  the  general  structure 
of  the  gland  is  seen  to  be  completely  altered.     The  distinction 


8 


»  a 


H  Oq       %      4a 


^m  ?*     ©%®®^<s^m    J? 


S 


0 


*9^&      ft0    ®         n  %^      6  -  ©*' 


0    0. 


^©0  <0       Q>       ®  Jfcv 


Fig.  31. — Lymphatic  Gland  in  Hodgkin's  Disease.     Drawn 
under  \  -inch  Objective. 

between  cortex  and  medulla  is  lost  and  the  germ  centres  have 
entirely  or  almost  entirely  disappeared.  The  gland  tissue  is 
composed  of  a  delicate,  but  obvious,  fibrous  reticulum  enclosing, 
comparatively  to  the  normal  gland,  scanty  cells.  Scattered 
throughout  the  gland  are  numerous  giant  cells,  fairly  obvious 
under  a  low  power. 

Under  the  J-inch  objective  the  cellular  content  of  the 
gland  is  seen  to  be  very  different  from  that  of  normal 
lymphatic  tissue.     Lymphoid  cells  are  present,  but  in  greatly 

p.  25 


386  CLINICAL   PATHOLOGY. 

diminished  numbers.  Endothelioid  cells  are  numerous.  The 
characteristic  giant  cells  of  Virchow  are  seen,  and  are  quite 
unlike  the  giant  cells  of  a  tuberculous  lesion.  The  Vii-chow 
cells  are  more  or  less  oval,  the  cytoplasm  resembling  that 
of  the  endothelioid  cells,  but  covering  a  considerably  wider 
area.  The  cells  are  multinucleated,  and  commonly  contain 
2,  4,  or  6  nuclei.  The  nuclei  are  placed  in  the  centre 
of  the  cells,  and  are  arranged  in  a  ring,  so  that  commonly 
2  nuclei  only  are  in  focus  at  one  time.  The  cells  are  some 
two  or  three  times  the  size  of  an  endothelioid  cell,  and  are  con- 
siderably smaller  than  the  large  tuberculous  giant  cells.  In 
addition  to  the  giant  cells  of  Virchow  a  large  number  of 
eosinophil  cells  are  nearly  always  present,  and  are  fairly  obvious 
in  the  ordinary  hematoxylin  and  eosin  sections.  To  display 
the  eosinophil  cells  to  greater  advantage  sections  may  be 
stained  by  Leishman's  stain  (page  433).  The  relative  prepon- 
derance of  cells  and  fibrous  tissue  differs  in  various  glands. 
The  fibrous  reticulum  may  be  greatly  increased,  producing  a 
tough  fibrous  gland ;  or  only  a  fine  fibrous  network  may  be 
present  with  a  soft  and  cellular  gland. 

The  main  points  to  be  looked  for  in  the  histological  diagnosis 
of  Hodgkin's  disease  are  : — 

The  replacement  of  the  normal  gland  structure  by  a  fibrous 
reticulum  containing  few  lymphoid  cells,  many  endothelioid 
cells,  eosinophils,  and  the  typical  multinucleated  cells  of 
Virchow. 

Molluscum  contagiosum. — Molluscum  contagiosum  is  a 
not  uncommon  condition.  It  occurs  in  the  form  of  small 
umbilicated  nodules  in  the  skin,  is  definitely  contagious,  and 
doubtless  of  parasitic  origin.  Molluscum  contagiosum  may  be 
found  in  any  part  of  the  skin,  and  may  be  inoculated  from  one 
part  to  another.  The  tumours  may  develop,  though  rarely,  on 
the  penis,  and  be  mistaken  for  chancres. 

Sections  through  a  nodule  have  a  very  characteristic 
appearance.  Under  the  low  power  the  nodule  is  seen  to  con- 
sist of  a  number  of  more  or  less  wedge-shaped  lobules  sejwated 
from  each  other  by  thin  fibrous  septa.  Each  lobule  has  an 
epithelial  lining  enclosing  round  or  oval  epithelial  cells.  The 
enclosed  cells  are  more  or  less  degenerated,  and  in  their  most 
advanced  state  become  swollen  homogeneous  masses  known  as 
molluscum  bodies.     The  molluscum  cells  have  been  considered 


THE   EXAMINATION   OF    SECTIONS,   ETC.      387 

to  be  psorosperms,  but  the  present  view  is  that  they  are 
derived  from  the  prickle  cells  of  the  skin  and  have  become 
keratinised. 


Fig.  32. — Molluscum  Contagiosum.     Drawn 
under  §-inch  Objective. 


The  Degenerations. 

Some  of  the  commoner  degenerations  are  not  infrequently 
met  with  in  tissues  removed  during  life ;  others  are  more  often 
studied  in  the  post-mortem  room.  The  clinical  pathologist 
may,  however,  meet  with  almost  any  pathological  tissue,  and 
the  more  important  degenerations  may  be  briefly  recorded 
here. 

Cloudy  swelling. — This  is  an  extremely  common  condi- 
tion met  with  in  inflammatory  processes  and  in  fevers.  The 
epithelial  and  connective  tissue  cells  are  affected.  In  the 
earlier  stages  the  cells  become  swollen,  lose  their  outline,  and 
stain  poorly ;  later  they  become  granular.  The  nuclei  stain 
feebly,  and  may  ultimately  disappear. 

(Edema. — The  appearance  of  the  cells  in  oedematous  tissues 
is  frequently  misleading  if  the  condition  is  not  recognised. 
The  cells  stain  badly,  and  become  greatly  distended  with  fluid* 
and  misshapen.  Cells  oedematous  but  otherwise  normal  are 
apt  to  be  mistaken  for  the  large  irregular  cells  of  a  carcinoma. 

Mucoid  degeneration. — This  form  of  degeneration  is 
similar  to  the  normal  process  met  with  in  mucous  tissues.  It 
occurs  also  in  catarrhal  conditions  and  in  some  forms  of  new 
growth 

25—2 


388  CLINICAL   PATHOLOGY. 

Mucoid  degeneration  is  found  in  some  varieties  of  sarcoma, 
and  particularly  in  the  myxo-sarcoma  met  with  in  the  antrum 
of  Highmore  and  other  parts  of  the  body.  Carcinomata  may 
also  show  the  change,  which  is  fairly  frequently  met  with  in 
carcinomata  connected  with  the  intestine. 

Sections  of  tissues  with  mucoid  degeneration  are  often 
difficult  to  prepare,  owing  to  the  fact  that  the  mucoid  material 
swells  up  when  the  sections  are  floated  in  water.  The  mucin 
is  found  both  in  the  cells  and  in  the  connective  tissue  fibres. 
The  substance  gives  the  reactions  of  mucin,  being  soluble  in 
dilute  alkalies  and  precipitated  by  acetic  acid. 

Sections  showing  mucoid  degeneration  should  be  stained 
with  carbol-thionin  or  van  Gieson's  stain  (page  481).  The 
mucin  stains  a  reddish  purple  colour  with  carbol-thionin. 

Colloid  degeneration. — Colloid  is  normally  met  with  in 
the  thyroid  gland,  and  in  the  anterior  glandular  part  of  the 
pituitary  body.  Colloid,  or  a  very  similar  substance,  may 
occur  as  a  result  of  degeneration  in  some  new  growths.  It 
is  found  in  the  secondary  deposits  of  thyroid  carcinoma  and 
in  some  ovarian  tumours. 

Colloid,  like  mucin,  swells  up  in  water,  but  is  not  precipitated 
by  acetic  acid.  It  is  often  brittle  and  difficult  to  cut  in  paraffin, 
but  sections  are  readily  made  by  the  gum  method. 

In  sections  stained  by  van  Gieson's  stain  colloid  is  a  bright 
orange-red. 

Hyaline  degeneration. — Hyaline  is  mainly  found  in  the 
adventitia  of  the  smaller  arteries  and  in  the  connective  tissues. 
The  connective  tissue  fibres  become  swollen  and  semi-trans- 
lucent. The  true  hyaline  change  is  probably  not  a  common 
one.  Van  Gieson's  stain,  which  gives  a  reddish  orange  with 
colloid  and  a  bright  rose-red  with  mucoid,  stains  hyaline  a 
flesh-pink  colour.  It  must  be  confessed,  however,  that 
absolute  differentiation  of  these  substances  by  van  Gieson's 
stain  is  far  from  certain. 

Lardaceous  degeneration. — It  is  not  definitely  settled 
whether  the  lardaceous  change  is  a  degeneration  of  the  tissue 
cells  or  an  infiltration  of  them  by  lardacein,  or  is  due  to  some 
other  substance  which  ultimately  becomes  lardacein.  The 
change  is  practically  confined  to  the  middle  coats  of  the  smaller 
arteries.  Lardaceous  change  may  follow  chronic  suppuration, 
or  syphilis,  or  both. 


THE   EXAMINATION   OF    SECTIONS,   ETC.      389 

The  change  is  not  commonly  met  with  in  tissues  removed 
during  life,  and  the  material  may  be  recognised  in  paraffin 
sections  by  its  staining  reaction  with  aniline  gentian-violet. 
The  section  can  be  stained  as  in  the  first  part  of  Gram's 
method  (page  432),  differentiated  in  saturated  oxalic  acid 
solution,  washed  in  water,  and  mounted  in  glycerine.  The 
lardacein  stains  a  bright  rose-red  and  the  tissues  bluish. 

Fatty  change. — Excess  of  fat  may  be  found  either  as  an 
increase  of  fat  in  normal  situations,  or  as  a  deposition  of  fat  in 
cells  which  do  not  normally  contain  it.  Both  fatty  infiltration 
and  fatty  degeneration  may  occur  together,  the  latter  condition 
being  the  more  important.  In  acute  fatty  degeneration  the 
globules  of  fat  are  as  a  rule  very  minute.  In  the  more  chronic 
forms  of  fatty  degeneration  the  globules  are  commonly  large, 
and  similar  to  those  found  in  fatty  infiltration  as  well  as  in 
normal  fatty  tissues. 

In  the  ordinary  paraffin  preparations  the  fat  is  dissolved 
out  in  the  process  of  passing  the  tissue  through  alcohol,  and 
the  areas  of  the  section  previously  occupied  by  globules  of  fat 
show  as  more  or  less  circular  empty  spaces.  The  finer  fatty 
changes  cannot  be  recognised  at  all  in  such  sections. 

To  demonstrate  fat  in  section  the  tissues  must  be  treated  by 
the  gum  process  and  the  sections  stained  by  one  of  the  fat 
stains,  such  as  Scharlach  K.  (page  425).  Scharlach  R.  stains 
fat  a  bright  red  colour  and  leaves  all  other  tissues  unstained. 
The  section  may  be  subsequently  counterstained  with  haema- 
toxylin,  washed  in  water,  and  mounted  in  Earrant's  solution. 

Calcareous  degeneration. — A  deposition  in  pathological 
tissues  of  calcium  carbonate  and  phosphate  is  not  uncommon 
in  various  conditions,  and  particularly  in  necrotic  tissues, 
tuberculous  foci,  and  in  some  malignant  tumours.  The  main 
interest  of  the  change  to  the  clinical  pathologist  lies  in  the 
damage  done  to  the  section  razor  by  the  small  gritty  particles, 
and  it  may  be  necessary  to  decalcify  such  tissues. 

The  calcareous  nodules  in  hematoxylin  and  eosin  sections 
stain  a  bluish  colour. 

Pigmentation. — Excess  of  pigment  in  tissues  may  arise 
from  a  variety  of  causes.  Extraneous  pigment  is  frequently 
found  in  cutaneous  tumours,  and  the  pigment  granules  may  be 
carried  deeply  into  the  tissues  and  into  the  neighbouring  lymph 
glands  by  the  wandering  cells.     Certain  new  growths,  and 


390  CLINICAL   PATHOLOGY. 

in  particular  melanotic  sarcomata,  are  deeply  pigmented,  and 
the  brownish  black  particles  are  seen  in  large  numbers  mainly 
within  the  cells  of  the  growth.  Hematogenous  pigmenta- 
tion occurs  as  the  result  of  local  bleeding  and  in  the  numerous 
general  conditions  associated  with  abnormal  blood  destruction. 
Pigment  derived  from  the  blood  is  often  of  a  golden  brown 
colour  in  section,  and  gives  the  free  iron  reaction  (pag6  433). 


CHAPTER  XXVII. 


NEOPLASMS — SIMPLE    TUMOUKS. 


The  neoplasms  are  conveniently  divided  into  "  simple  " 
and  "malignant"  tumours.  Each  division  can  be  sub- 
divided into  tumours  of  epithelial  or  glandular  structure, 
and  tumours  of  the  connective  tissues.  Only  those  tumours 
which  are  frequently  or  comparatively  frequently  met  with 
can  be  mentioned  here. 

Simple  Tumours. 

Papilloma. — Papillomata  are  epithelial  tumours,  and 
consist  of  localised  out-growths  of  epithelial  surfaces.  The 
cells  forming  the  tumours  are  squamous  or  columnar, 
according  to  the  nature  of  the  epithelium  from  which  they 
are  growing.  Papillomata  are  often  multiple,  commonly 
arise  as  the  result  of  a  local  irritant,  and  can  in  some 
cases  be  inoculated  by  contact  upon  neighbouring  tissues. 
Examples  of  inoculation  may  be  seen  in  papillomata  of  the 
gut,  the  bladder,  and  the  larynx. 

A  papilloma  of  the  skin  reproduces  the  structure  of  the 
normal  papillae,  and  if  exposed  to  friction  the  epidermal  cells 
multiply  above  the  papilla  forming  a  callosity,  as  in  a  corn. 
Increase  and  keratinisation  of  the  horny  cells  produces  a 
keratoma.  A  wart  is  a  typical  instance  of  a  cutaneous 
papilloma.  A  papilloma  of  a  mucous  membrane,  as  of  the 
bladder  or  intestine,  is  covered  with  a  few  layers  of  cells 
only,  and  being  well  supplied  with  blood-vessels  is  very  apt 
to  bleed. 

Papillomata  may  develop  also  from  the  epithelial  lining  of 
cysts,  and  are  particularly  common  in  ovarian  tumours.  The 
papillary  processes  involved  frequently  become  branched,  and 
the  resulting  tumour  is  consequently  dendritic.  Papillomata 
may  undoubtedly  become  malignant  and  are  then  converted  into 
(or  replaced  by)  carcinomata,  a  transformation  which  appears 
to  be  particularly  common  in  papillomata  of  the  bladder.     It 


392  CLINICAL   PATHOLOGY. 

is,  however,  most  desirable  to  distinguish  between  simple 
and  malignant  growths  of  this  nature.  In  dealing  with 
typical  warty  papillomata  of  the  skin  the  distinction  is  easy, 
but  in  the  case  of  bladder,  intestinal,  or  laryngeal  growths 
there  may  be  considerable  difficulty,  and  intermediate  stages 
in  the  conversion  of  a  papilloma  into  a  carcinoma  may  be 
encountered. 

The  papilloma  removed  should  have,  if  possible,  an  area  of 
the  surrounding  epithelium  and  a  portion  of  the  subjacent 
tissues  attached  to  it.  Great  care  should  be  taken  in  the 
"  casting  "  of  the  paraffin  block,  so  that  the  sections  are  cut 
exactly  at  right  angles  to  the  epithelial  surface.  If  a  section 
is  cut  in  a  slanting  direction  through  normal  skin  an 
appearance  of  epithelial  thickening  and  down-growth  is 
obtained,  which  is  confusing  to  the  beginner.  The  section 
should  be  examined  first  with  a  hand  glass,  when  the  deeply 
stained  line  of  epithelium  is  seen  projecting  towards  the 
surface  instead  of,  as  in  a  carcinoma,  dipping  down  into  the 
subjacent  tissues.  Under  the  microscope  the  epithelial 
outgrowth  is  seen  to  be  regular,  and  to  more  or  less  exactly 
reproduce  the  character  of  the  adjacent  epithelium.  The 
regularity  of  the  outgrowth,  the  comparatively  normal  size, 
shape  and  appearance  of  the  cells  composing  it,  and  the 
absence  of  epithelial  infiltration  deeply  into  the  subjacent 
tissues,  serve  to  distinguish  the  simple  from  the  malignant 
tissue. 

Adenoma. — Adenomata  are  somewhat  similar  tumours  to 
the  foregoing,  but  arise  from  glandular  structures  instead  of 
from  epidermal  or  mucous  surfaces.  They  tend  to  repro- 
duce the  type  of  glandular  tissue  from  which  they  develop, 
and  in  some  instances  reproduce  it  so  closely  that  on  purely 
histological  grounds  they  cannot  be  distinguished  from  the 
normal  gland  or  a  general  overgrowth  of  it.  An  adenoma 
of  the  thyroid  gland,  for  example,  under  the  microscope 
exactly  resembles  a  simple  hypertrophy  of  the  gland,  but  is 
readily  distinguished  by  its  gross  appearance,  since,  like  all 
adenomata,  it  is  a  localised  and  usually  encapsulated  tumour. 
Adenomata  arising  from  columnar-celled  glandular  tissue  have 
a  tubular  structure  lined  by  columnar-celled  epithelium.  The 
tubes  may  be  covered  with  connective  tissue,  or  the  tissue  may 
be  thrown  into  a  fold  producing  a  papillary  projection  lined 


NEOPLASMS-SIMPLE   TUMOURS. 


393 


by  epithelium.  Such  a  tumour  is  known  as  a  papillary- 
adenoma-  Adenomata  arising  from  spheroidal-celled  tissue, 
such  as  that  of  the  breast,  are  composed  of  similar  cells 
grouped  into  alveoli.  If  the  interstitial  fibrous  connective 
tissue  is  in  much  excess  the  tumours  are  called  fibro- 
adenomata-  Adenomata  are  met  with  in  the  breast,  the 
prostate,  the  uterus,  the  ovary,  the  intestine  (where  they  may 
form  pedunculated  growths  or  "polypi"),  the  sebaceous 
glands,   the   kidney,  and  the  gall  bladder.      The  adenomata 


'/ 


ft 

s 

G> 


l    % 


ff8   0 


V  •«  1)1 


/ 


/ 


fcb  °e 


•©    o 


Fig.  33. — Adenoma  of  Breast.     Drawn  under  §-inch  Objective. 


rarely    become  carcinomata,  and  have    to    be   distinguished 
from  them.     The  points  of  distinction  are  the  following  : — 

The  adenomata  are  capsulated  and  the  carcinomata  are 
very  rarely  capsulated.  The  adenoma  cells  do  not  infiltrate 
the  surrounding  tissues.  The  cells  of  an  adenoma  retain 
considerable  likeness  to  those  of  the  normal  tissues,  and  tend 
to  reproduce  a  more  or  less  regular  tubular  or  alveolar 
structure.  They  contain  as  a  rule  a  considerable  fibrous 
tissue  framework,  which  supports  the  tubular  or  acinous  cell 
collections,  but  is  not  invaded  by  them.  The  microscopic 
appearance  of  a  fibro-adenoma  conveys  the  impression  of  a 


394  CLINICAL   PATHOLOGY. 

fibrous  framework  supporting  a  series  of  glandular  processes ; 
the  appearance  of  a  scirrhus  or  fibro-carcinoma  is  that  of 
cellular  columns  invading  and  eating  their  way  into  a  fibrous 
barrier. 

In  distinguishing  between  an  adenoma  and  a  carcinoma  of 
the  uterus,  it  must  be  remembered  that  the  glands  of  the 
endometrium  normally  penetrate  a  considerable  distance 
below  the  surface.  The  same  is  true  of  glands  of  the  gall 
bladder. 

Lymphoma. —Lymphoma  is  the  name  applied  to  tumour- 
like masses  composed  of  lymphoid  cells  such  as  occur  in  the 
leukaemias  and  in  general  lymphoid  hyperplasia.  They  have 
a  very  close  histological  resemblance  to  round-celled  sarco- 
mata.    The  term  is  also  applied  to  a  lympho-sarcoma. 

Fibroma. — This  is  a  connective  tissue  tumour  composed 
mainly  of  fibrous  tissue,  and  may  occur  in  any  organ  contain- 
ing connective  tissue.  Fibromata  are  found  commonly  in  the 
skin,  fasciae,  periosteum,  tendons,  nerves,  uterus,  ovary,  breast, 
and  nose.  In  the  skin  the  pendulous  fibroma  may  cover  a  con- 
siderable area,  and  is  known  as  molluscum  fibrosum.  It  is, 
however,  more  properly  described  as  a  neuro -fibroma.  The 
common  "  fibroid"  tumour  of  the  uterus  is  largely  made  up 
of  unstriped  muscle,  and  is  known  as  a  leiomyoma.  The 
epulis  is  often  a  fibroma  and  less  commonly  a  myeloid 
sarcoma.  The  "mucous  polyp  "  of  tbe  nose  consists  of  a 
loose  cedematous  connective  tissue  framework  enclosing  a 
number  of  inflammatory  cells.  It  is  commonly  regarded  as 
an  cedematous  fibroma.  Fibromata  may  be  hard  or  soft, 
according  to  the  density  of  the  fibrous  tissue  and  the  presence 
or  absence  of  oedema  and  mucoid  degeneration  to  which  these 
tumours  are  occasionally  liable. 

Under  the  microscope  the  fibroma  is  readily  recognised  by 
its  elongated  spindle-shaped  and  wavy  cells,  which  tend  to  be 
arranged  in  whorls.  Blood-vessels  are  usually  scanty,  but 
well  formed. 

Many  of  the  fibromata  contain  other  elements  than  fibrous 
tissue,  and  a  certain  admixture  of  elastic  tissue  is  usual. 
Some  fibromata  contain  also  unstriped  muscle  and  are  known 
as  fibro-myomata,  or  if  the  muscle  tissue  predominates  as 
leiomyomata.  Others  contain  also  nerve  tissue  and  are  called 
fibro-neuromata,  and  the  name  fibre-adenoma  is  applied 


NEOPLASMS— SIMPLE   TUMOURS.  395 

to  an  adenoma,  such  as  is  commonly  met  with  in  the  breast, 
and  contains  little  glandular  and  much  fibrous  tissue. 
Degenerated  fibromata  and  leiomyomata  are  often  difficult  to 
distinguish  from  spindle-celled  sarcoma,  and  a  sarcomatous 
change  may  occur  in  the  simple  tumour.  The  regular  arrange- 
ment of  mature  long  fibrous  tissue  cells  arranged  in  whorls  is  not 
seen  in  sarcoma.  Moreover  the  few  vessels  of  a  fibroma  are 
usually  thick-walled,  while  the  rather  more  numerous  blood- 
vessels of  a  sarcoma  are  mere  clefts  in  the  tissue  containing 
blood,  and  lined  as  a  rule  by  a  single  layer  of  cells. 


~~p/r^^i_z. ""  .-::-=-.-_- -^ ~      -W:T"S5: 

/^^2g|f=^  ^  ^  v^^^r^^pr^  j . 

~M 

?•  ~z^P^^~£Sk "  ^^  •  "\^ N  \  i '  !i  1 , "'  1 4> '  '^sss^ 

S?5?^^^. 

Q^^S&^W&S 

">s<^ 

'f.of     v    N     %S 

Pi 

/"^'iS^!^^?:^^^  -"  '^ 

yV  .,  ~^  £"--.':  " 

:  v^^^S^ 

Fig.  34. — Fibroma  of  Ovary.     Drawn  under  §-inch  Objective. 

Myoma. — Myomata  may  be  composed  of  striated  or  of 
unstriated  muscle  fibres.  The  former  class  of  tissue  is  called 
a  rhabdomyoma,  and  is  practically  unknown.  The  rhabdo- 
myoma of  the  kidney  is  generally  considered  to  be  a  sarcoma. 

Leiomyoma  is  composed  of  unstriped  muscle  fibre,  and  is 
the  common  tumour  of  the  uterus  known  as  the  "  fibroid." 
The  leiomyomata  of  the  uterus  usually  contain  a  varying 
amount  of  fibrous  tissue,  and  may  properly  be  called  fibro- 
myomata.  Similar  tumours  are  also  found  in  the  ovary  and 
less  commonly  in  the  prostate. 

Under  the  microscope  the  arrangement  of  the  muscle  cells 
in  whorls  is  very  similar  to  the  arrangement  of  the  fibrous 
tissue  in  a  fibroma,  and  a  similar  distinction  in  the  case  of 
cedematous  or  degenerated  growths  has  to  be  made  between 


396  CLINICAL   PATHOLOGY. 

myoniata  and  sarcomata.  The  myoniata  usually  contain  few, 
but  well-formed,  blood-vessels.  The  tendency  of  uterine  fibroids 
to  cause  bleeding  from  the  uterus  results  from  their  situation. 
The  haemorrhages  come  from  the  closely-subjacent  endo- 
metrium, and  not  from  the  tumour. 

Myxoma. — The  true  myxoma  is  a  rare  tumour  composed 
of  similar  tissue  to  the  Whartonian  jelly  of  the  umbilical  cord. 
The  myxomata  may  form  tumours  of  considerable  size  in  the 
subcutaneous  tissues,  and  occur  also  in  the  medullary  cavities 
of  the  long  bones.  They  are  frequently  mixed  tumours, 
myxo-liioomata,  fibro-myxomata,  chondro-myxomata,  and 
myxo-sarcomata  being  met  with. 

The  tumour  is  seen  to  consist  of  a  mucinous  tissue  contain- 
ing spider-like  cells  with  a  darkly-staining  nucleus  and  a 
considerable  amount  of  cytoplasm,  the  long  interlacing  pro- 
cesses of  the  cells  forming  the  framework  of  the  tissue.  The 
cells  are  separated  by  considerable  intervals. 

Lipoma. — The  .lipomata  are  among  the  commonest  and 
most  readily  recognised  of  all  the  tumours.  They  are  found 
in  almost  any  situation  where  fatty  tissue  is  normally 
abundant,  such  as  the  neck,  shoulders,  axillae,  and  groins. 
They  may  be  circumscribed  and  lobulated,  forming  more  or 
les3  pedunculated  tumours,  which  may  reach  a  considerable 
size,  or  they  may  be  diffuse  and  consist  of  a  relatively  localised 
overgrowth  of  the  tissue  fat.  Under  the  microscope  liporoata 
are  seen  to  consist  of  fatty  tissue  with  a  varying  framework  of 
fibrous  tissue.  In  paraffin  sections  they  appear  as  a  loose  and 
empty  reticulum  of  fibrous  tissue. 

Chondroma. — Cartilaginous  tumours  are  of  two  varieties. 
The  variety  which  grows  from  normally  situated  cartilage  is 
known  as  an  ecchondroma.  Those  arising  in  situations 
where  cartilage  is  normally  absent  are  called  enchondromata. 
Ecchondromata  are  usually  small  tumours  arising  from  the 
cartilage  of  the  ribs,  the  larynx,  or  the  nasal  septum.  The 
enchondromata  are  usually  attached  to  the  long  bones,  and 
particularly  to  the  phalanges.  Cartilage  may  also  be  found  in 
parotid,  testis,  and  kidney  tumours.  The  cartilage  found 
in  parotid  tumours  is  supposed  to  be  derived  from  Meckel's 
cartilage  of  the  bronchial  arches.  The  enchondromata  are 
often  multiple,  and  may  become  sarcomatous  and  form 
secondary  deposits. 


NEOPLASMS— SIMPLE    TUMOURS.  397 

Under  the  microscope  chondromata  are  seen  to  consist  of 
hyaline  cartilage  containing  a  variable  number  of  unevenly 
distributed  cartilage  cells.  In  some  cases  the  matrix  is  not 
homogeneous,  but  fibrillated  like  that  of  fibro-cartilage. 

Osteoma. — By  osteoma  is  meant  a  tumour  composed  of 
bone,  and  not  a  bony  overgrowth,  such  as  is  found  in  the  callus 
which  surrounds  a  fracture.  Osteomata  of  two  kinds  are 
recognised.  The  cancellous  osteoma  is  composed  of  bony 
trabecule  including  large  irregular  spaces,  a  considerable 
amount  of  medulla,  and  a  number  of  blood-vessels.  The  ivory 
osteoma  is  composed  of  dense  bone,  with  little  medulla  and 
few  vessels.  Osteomata  of  the  teeth  composed  of  dentine  are 
known  as  odontomata,  and  those  composed  of  cement  as 
dental  osteomata.  Osteomata  are  frequently  multiple,  and 
their  most  usual  situations  are  the  skull,  the  extremities  of 
the  long  bones,  and  the  pelvis.  Ivory  osteomata  are  practi- 
cally confined  to  the  skull. 

Angeioma. — The  angeiomata  are  tumours  composed  of 
vessels  which  may  be  either  blood  or  lymph  channels.  The 
former  class  of  tumour  is  called  a  hsemangeioma,  the  latter 
a  lymphang-eioma. 

The  hpemangeiomata  are  further  subdivided  into  capillary 
or  simple  angeiomata,  and  into  venous  or  cavernous 
angeiomata. 

Both  classes  of  haemangeionia  are  also  known  as  nffivi. 
Capillary  nsevi  contain  large  numbers  of  tortuous,  distended, 
and  thick-walled  capillaries  lined  by  a  well-marked  layer  of 
endothelial  cells  and  lying  in  ordinary  connective  tissue.  They 
are  congenital  tumours  for  the  most  part,  and  often  tend  to 
increase  in  size.  They  are  very  obvious  during  life,  and  may 
be  much  less  distinctive  in  a  microscopic  section.  If  the 
tissue  has  been  fixed  with  the  vessels  distended  by  blood  the 
histological  aspect  of  an  area  composed  of  many  small 
vessels  containing  red  cells  is  unmistakable.  If,  however,  as 
not  infrequently  happens,  the  vessels  have  contracted  their 
fibrous  walls  and  emptied  their  lumina  of  blood,  the  section 
has  rather  the  appearance  of  ordinary  connective  tissue. 

Capillary  angeiomata  may  occur  on  any  part  of  the  body, 
but  are  particularly  common  on  the  face.  They  may  be 
single  or  multiple. 

Venous  or  cavernous  angeiomata  consist  merely  of  wide, 


398  CLINICAL  PATHOLOGY. 

irregular  clefts  lined  with  endothelium  and  separated  by 
fibrous  septa.  They  occur  on  the  skin,  forming  the  raised 
purplish  patches  known  as  naevi  or  birth  marks.  They  are 
found  occasionally  in  the  internal  organs,  but  very  rarely  in 
any  other  organ  than  the  liver,  where  they  are  not  particularly 
uncommon. 

Lymphangeiomata  are  rare  tumours  consisting  of  collec- 
tions of  dilated  lymph-vessels.  Two  varieties  are  described. 
The  cavernous  lymphangeiomata  occur  in  the  tongue  and  the 
lips,  and  are  concerned  in  the  production  of  macrogiossia  and 
macrocheilia.  Cystic  lymphangeiomata  are  rare  tumours  which 
occur  in  the  subcutaneous  tissues,  and  particularly  those  of  the 
neck,  forming  diffuse,  cystic,  and  fluctuating  swellings.  The 
lymphangeioma  spaces  are  recognised  under  the  microscope 
by  their  being  filled  with  a  homogeneous  watery  lymph,  and 
by  the  absence  of  red  cells  in  them. 

Moles. — Moles  are  usually  pigmented  and  often  hairy 
tumours  growing  in  the  skin.  Some  confusion  in  the  nomen- 
clature is  caused  owing  to  the  custom  of  German  authors  in 
naming  these  growths  naevi  and  of  English  authors  for  the 
most  part  calling  the  common  capillary  angeiomata  of  the  skin 
nsevi.  The  pigmented  and  often  hairy  growths  described  here 
as  moles  may  be  highly  vascular,  but  are  histologically  and 
clinically  quite  distinct  from  the  capillary  angeiomata. 

Apart  from  the  confusion  of  nomenclature  the  classification 
of  these  tumours  is  sufficiently  difficult.  They  are  considered 
by  some  authors  as  epithelial,  by  others  as  of  mesoblastic 
origin.  The  simple  moles  have  a  tendency  to  become 
malignant,  and  may  give  rise  to  the  most  intensely  malignant 
of  all  tumours,  namely,  the  melanotic  sarcoma.  It  is  by  no 
means  easy  in  all  cases  to  judge  on  histological  grounds 
whether  a  mole  is  simple  or  malignant,  and  it  is  particularly 
necessary  that  a  mole,  if  removed,  should  be  taken  away  with 
a  wide  margin  and  a  portion  of  the  deeper  structures,  both 
to  avoid  recurrence,  and  to  enable  a  proper  histological 
examination  to  be  made.  The  majority  of  moles  are  composed 
of  epithelioid  cells  in  an  alveolar  arrangement.  Prolongations 
of  the  overlying  epithelium  downwards  help  to  form  the 
alveolar  network,  within  the  meshes  of  which  are  found  groups 
of  the  somewhat  angular  epithelioid  cells.  The  cells  are 
divided  into  groups  by  fibrous  tissue,  but  the  stroma  does  not 


NEOPLASMS— SIMPLE   TUMOURS.  399 

pass  between  the  cells.  The  great  majority  of  the  tumours 
contain  a  large  number  of  brownish-black  pigment  granules. 
The  pigment  as  a  rale  is  most  abundant  towards  the  surface 
of  the  growth. 

Blood-vessels  may  be  numerous,  and  small  cysts  may 
develop  within  the  tumours. 

Even  in  the  non-malignant  moles  the  extension  of  the 
tumour  downwards  beneath  the  epithelium  may  be  consider- 
able, and  the  margin  of  the  growth  is  often  ill  defined  and 
irregular.  There  is  no  capsule.  The  extension  of  the  growth 
through  the  subcutaneous  loose  connective  tissue  into  the 
fasciae  and  muscles  is  evidence  of  malignancy,  as  is  also  the 
replacement  of  the  compact  alveolar  arrangement  by  a  looser 
and  more  irregular  cell  growth. 

Moles  are  found  on  almost  any  part  of  the  skin  and  on 
the  conjunctiva.  They  are  congenital  tumours,  and  if  pig- 
mented arise  only  in  situations  where  pigment  is  normally 
present. 

Glioma. — Gliomata  are  comparatively  rare  tumours,  the 
nature  and  classification  of  which  are  considerably  confused. 
They  can  hardly  be  considered  simple  tumours,  since  they 
infiltrate  the  surrounding  tissues,  yet  they  are  usually 
classified  as  such.  The  gliomata  are  essentially  tumours  of 
the  central  nervous  system,  and  as  such  are  confined  to  the 
brain  and  the  spinal  cord.  The  gliomata  of  the  retina  are 
customarily  grouped  with  those  of  the  brain  and  cord,  but 
are  in  reality  tumours  of  a  very  different  nature,  and  are 
mentioned  here  only  in  obedience  to  the  usual  custom.  The 
gliomata  of  the  brain  and  cord  are  of  two  kinds.  They  may 
form  definite  and  more  or  less  encapsulated  tumours,  which 
may  be  single  or  multiple,  or  they  may  be  indefinite  infiltra- 
tions the  edges  of  which  cannot  be  determined  by  the  naked 
eye.  The  diffuse  gliomata  tend  to  soften  and  break  down  and 
to  destroy  the  neighbouring  tissues.  The  condition  of  the 
cord  producing  the  disease  syringo-myelia  is  considered  to 
result  from  a  gliomatous  growth.  The  gliomata  are  usually 
slowly-growing  tumours,  and  occur  in  adult  life.  Under  the 
microscope  the  gliomata  are  seen  to  consist  of  glial  cells, 
which  contain  a  relatively  small  amount  of  cytoplasm  and 
have  delicate  branching  processes  which  divide  and  interlace 
in  all  directions.     The  nuclei  are  relatively  large  and  stain 


400  CLINICAL   PATHOLOGY. 

deeply.  The  number  of  blood-vessels  in  the  tumour  varies 
considerably,  as  also  do  the  nerve  fibres  and  cells  which  may 
be  incorporated  in  it.  This  variety  of  glioma,  consisting  almost 
entirely  of  mature  glial  tissue,  is  not  met  with  in  the  retina. 

The  retinal  glioma  differs  markedly  both  on  clinical  and  on 
histological  grounds  from  those  found  in  the  brain  and  cord. 
It  is  met  with  in  babies  or  very  young  children  and  is 
intensely  malignant,  growing  with  great  rapidity  and  killing 
by  invasion  of  neighbouring  structures.  It  may  commence 
simultaneously  in  both  eyes.  Several  members  of  the  same 
family  may  be  affected. 

Under  the  microscope  the  tumour  is  seen  to  consist  of  an 
embryonic  type  of  tissue  in  which  all  the  structures  of  the 
retina  have  been  traced.  Eods  and  cones  may  be  found.  The 
cells  have  few  or  none  of  the  delicate  processes  seen  in  the 
gliomata  of  the  brain.  The  best-formed  cells  are  seen  growing 
round  the  vessels  in  small  masses,  which  are  separated  by 
non-vascular  areas  of  necrosed  cells. 

It  is  evident  that  a  retinal  glioma  can  hardly  be  described 
as  a  simple  tumour  and  with  difficulty  as  a  sarcoma,  since  all 
the  retinal  structures  may  be  repeated  in  it.  It  might, 
appropriately,  be  called  a  retinal  "neuro-blastoma." 

Neuroma. — Neuromata  are  tumours  partly  composed  of 
nerve  fibres  or  cells.  The  conditions  to  which  the  term 
"neuroma"  should  be  applied  are  in  considerable  confusion 
owing  to  the  disputed  character  of  the  growths.  Some  have 
been  considered  to  be  fibromata  which  have  merely  entangled 
nerve  fibres  in  them,  but  the  general  tendency  is  to  call  those 
tumours  which  contain  nerve  fibres  or  cells  neuromata  or 
neuro-fibromata.  The  tumours  are  rare  and  can  only  be 
referred  to  here.  Molluscum  fibrosum  has  already  been 
briefly  considered,  and,  since  the  cutaneous  growths  tend  to 
follow  the  lines  of  nerve  supply  and  contain  nerve  fibres,  they 
are  generally  classed  as  neuro-fibromata. 

Single  tumours  may  also  arise  in  nerve  sheaths  and  may 
apparently  be  composed  purely  of  fibrous  tissue  derived  from 
the  nerve  sheath,  or  more  commonly  contain  nerve  fibres  in 
them.  The  rare  condition  known  as  plexiform  neuroma  is  a 
multiple  growth  affecting  one  or  more  systems  of  nerves,  and 
associated  with  great  thickening  of  the  nerve  sheaths  and  a 
formation  of  new  and  often  abnormal  nerve  fibres. 


NEOPLASMS- SIMPLE    TUMOUES.  401 

Endothelioma. — The  endothelioma  is  a  comparatively  rare 
tumour  of  uncertain  classification.  It  is  commonly  classed 
among  the  sarcomata  because  of  its  mesoblastic  origin,  but  in 
its  histological  characters  it  resembles  rather  the  carcinomata 
or  papillomata,  and  from  its  clinical  characters,  since  it  has 
little  tendency  to  infiltrate  the  surrounding  tissues  or  to  form 
metastases,  it  might  well  be  classed  as  a  simple  tumour. 
Numerous  other  tumours  difficult  to  classify,  such  as  the 
mixed  tumour  of  the  parotid,  are  for  no  very  good  reasons 
included  among  the  endotheliomata. 

The  endothelioma  arises  from  endothelium,  and  is  mainly 
composed  of  endothelial  cells.  It  is  found  growing  from  the 
membranes  of  the  brain  and  less  commonly  from  the  pleura, 
or  peritoneum.  Under  the  microscope  the  tumours  are  seen 
to  consist  of  collections  of  large  endothelial  cells  arranged 
in  groups,  some  of  which  show  a  whorled  arrangement. 
The  individual  cells  are  not  separated  from  each  other, 
as  in  the  sarcomata,  by  connective  tissue,  but  are  in 
apposition. 

Tumours  composed  of  cells  arranged  in  columns  the 
centres  of  which  have  undergone  degeneration  are  known  as 
cylindromata. 

Endothelial  tumours  arising  from  the  dura  and  containing 
a  considerable  amount  of  fibrous  tissue  together  with  calcareous 
granules  are  called  psammomata. 

The  parotid  tumour. — A  peculiar  form  of  "mixed" 
tumour,  known  as  the  parotid  tumour,  is  not  infrequent  in 
the  parotid  gland,  and  may  occur  in  the  other  salivary 
glands. 

The  tumour  is  a  very  distinctive  and  remarkable  one,  which 
has  been  variously  classified  as  an  endothelioma,  a  chondroma, 
a  sarcoma,  an  adenoma,  etc.  It  is  perhaps  wisely  described 
as  a  "  mixed  "  tumour. 

The  parotid  tumour  is  localised  and  encapsulated,  and, 
though  it  has  a  distinct  tendency  to  recur  after  removal,  does 
not  disseminate,  and  is  conveniently  classed  here  among  the 
simple  tumours. 

Under  the  microscope  are  seen  strands,  columns,  and  often 
whorls  of  small,  somewhat  angular  and  elongated  cells  with 
deeply -staining  nuclei.  The  cellular  portions  are  often 
separated    by    considerable   areas    of   a    hyaline   groundwork 

p.  26 


402  CLINICAL   PATHOLOGY. 

containing  few  and  small  groups  of  cells  only.  Occasional 
fairly  well  defined  areas  of  cartilaginous  material  are  met 
with,  which  are  said  to  arise  from  inclusions  of  Meckel's 
cartilage.  The  cells  in  the  cartilage  are  usually  scanty  and 
their  arrangement  is  atypical.  Portions  of  the  stroma  may 
be  mixed  with  fibrous  tissue,  which  in  other  parts  forms  the 
hyaline  groundwork  previously  mentioned. 

The  tumours  may  reach  a  considerable  size,  and  are  not 
always  wholly  encapsulated.  Very  rarely  they  may  grow 
into  neighbouring  glands,  but  dissemination,  as  previously 
stated,  does  not  seem  to  occur. 

Relationship  between  the  simple  and  malignant 
tumours. — Many  of  the  simple  tumours  may  become  malig- 
nant, invade  neighbouring  tissues,  and  disseminate.  This 
conversion  of  a  simple  into  a  malignant  tumour  is  a  different 
process,  and  can  often  be  distinguished  histologically  from 
the  invasion  of  a  simple  tumour  by  a  malignant  one.  A 
glandular  carcinoma,  for  example,  can  be  seen  to  infiltrate  the 
very  different  structure  of  an  old-standing  parotid  tumour. 
If  a  simple  tumour  becomes  malignant  it  necessarily  can  only 
take  on  one  form  of  malignant  growth  structure,  namely,  the 
form  which  arises  spontaneously  in  the  tissue  of  which  the 
simple  tumour  is  made  and  from  which  it  grows.  Thus  a 
papilloma  of  the  skin  becomes  an  epithelioma  or  squamous- 
celled  carcinoma  :  an  adenoma  becomes  a  glandular  carcinoma  : 
a  fibroma  becomes  a  spindle-celled  sarcoma.  The  great 
majority  of  malignant  growths,  however,  arise  from  normal 
tissues  and  not  from  simple  tumours. 


CHAPTEE   XXVIII. 

CARCINOMATA — SARCOMATA — OTHER    TUMOURS — CYSTS. 

Malignant  tumours  differ  from  simple  neoplasms  in  that  they 
have  no  well-defined  edge  and  no  capsule ;  that  they  grow  rapidly 
and  invade  neighbouring  structures  ;  that  they  form  multiple 
secondary  growths  of  the  same  nature  at  a  distance  ;  and  that 
they  commonly  produce  cachexia.  All  malignant  growths, 
however,  do  not  conform  to  these  characteristics. 

The  malignant  tumours  are  divided  into  two  main  groups  : 
tumours  arising  from  epiblast  or  hypoblast  are  known  as 
carcinomata ;  malignant  connective  tissue  tumours  arising 
from  mesoblast  are  known  as  sarcomata.  The  division  is  thus 
analagous  to  that  made  between  the  simple  neoplasms. 

Carcinomata  differ  from  sarcomata  in  several  respects,  and  in 
the  great  majority  of  instances  are  readily  distinguished  from 
them. 

Sarcomata  have  many  thin-walled  blood-vessels,  but  no 
lymphatics.  A  delicate  connective  tissue  penetrates  between 
each  cell,  but  may  be  so  delicate  as  to  be  discerned  only  with 
great  difficulty.  The  tumours  are  very  cellular,  and  the 
arrangement  of  the  cells  is  commonly  irregular.  Dissemina- 
tion takes  place  by  the  blood-stream  rather  than  by  the 
lymphatics.  The  tumours  may  arise  at  any  age,  but  are  most 
common  in  young  subjects. 

Carcinomata  possess  a  stroma  carrying  more  or  less  wel]- 
formed  blood-vessels  and  lymphatics.  The  cells  of  the  growth 
are  in  apposition  to  each  other.  The  groups  of  cells  are 
arranged  in  columns  or  in  rude  alveoli.  Dissemination  takes 
place  by  the  lymphatics  rather  than  by  the  blood-stream.  The 
tumours  arise  most  commonly  in  middle-aged  and  elderly 
subjects. 

The  Carcinomata. 

Carcinomata  tend  to  reproduce  the  structure  of  the  tissue  in 
which  they  arise,  but  very  imperfectly.  The  cells  of  which 
they  are  composed  are  evidently  similar  to  the  cells  of  the 

26—2 


404  CLINICAL   PATHOLOGY. 

tissue  of  origin,  and  the  secondary  deposits,  in  whatever  organ 
they  occur,  are  composed  of  cells  similar  to  those  of  the 
primary  growth.  Yery  cellular  carcinomata  are  often  called 
"  encephaloid,"  and  tumours  with  comparatively  few  cells 
and  much  fibrous  tissue  are  known  as  "  scirrhous."  All 
forms  of  carcinoma  are  vulgarly  referred  to  as  cancers. 

Carcinomata  are  conveniently  divided,  according  to  the  type 
of  cell  of  which  they  are  composed,  into  squamous,  spheroidal 
or  cuboidal,  and  columnar  celled  carcinomata. 

The  term  "  epithelioma  "  is  applied  by  some  to  all  epithelial 
tumours,  and  to  avoid  confusion  is  not  used  here. 

Squamous-celled  carcinoma. — This  variety  of  carcinoma 
necessarily  arises  from  squamous  epithelium,  and  consequently 
is  found  upon  any  part  of  the  skin,  the  lips,  the  tongue  and 
buccal  cavity,  the  tonsil,  pharynx,  larynx  and  oesophagus,  the 
anus,  the  cervix  uteri,  the  glans  penis,  and  the  bladder. 
The  commonest  situations  are  the  cervix  uteri,  the  lip, 
and  the  tongue.  Squamous-celled  carcinoma  is  particularly 
liable  to  develop  in  response  to  chronic  irritation.  It 
rarely  occurs  upon  the  skin,  but  may  be  grafted  upon  X-ray 
burns,  upon  a  chronic  sore,  or  notoriously  upon  the  scrotum  of 
a  sweep. 

Carcinoma  of  the  lip  is  practically  confined  to  pipe  smokers, 
and  particularly  to  those  who  suck  clay  or  painted  horn  mouth- 
pieces, and  it  arises  at  the  place  on  the  lip  where  the  pipe  is 
habitually  held.  Carcinoma  of  the  tongue  is  rare  except 
among  patients  with  syphilitic  leukoplakia.  Carcinoma  of 
the  penis  is  practically  confined  to  the  uncircumcised. 

This  variety  of  carcinoma  is  locally  very  malignant,  and 
spreads  rapidly  into  the  neighbouring  glands.  It  rarely 
disseminates  generally  over  the  body. 

The  least  malignant  form  of  carcinoma  is  that  affecting  the 
lip.  It  attracts  notice  early  and  remains  localised  for  a  con- 
siderable time.  Operative  treatment  is  far  more  successful  in 
cases  of  carcinoma  of  the  lip  than  in  any  other  form  of 
carcinoma  with  the  exception  of  rodent  ulcer. 

The  histological  diagnosis  of  squamous-celled  carcinoma  is 
usually  easy.  Care  should  be  taken,  however,  to  cut  the 
sections  exactly  at  right  angles  to  the  epithelial  surface.  The 
only  growths  which  could  be  mistaken  for  this  form  of  carci- 
noma are  papillomata  and  rodent  ulcers. 


CARCINOMATA— SARCOMATA,   ETC. 


405 


Under  the  microscope  the  following  points  can  be  made  out : — 
An  ulcer  bare  of  epithelium  is  usually  present,  and  at  the 
edges  of  the  ulcer  the  epithelium  is  thickened  and  irregular, 
and  dips  down  into  the  subjacent  tissues.     Beneath  the  base 


Fig.  35. — Squamous- Celled  Carcinoma  of  Tongue.     Drawn 
under  f-inch  Objective. 


"S7 


&   ®    O 


flir^wf  »  ss 


0  9    (V 


Fig.  36. — CelL  Nest.     Drawn  under  £-mch  Objective. 

of  the  ulcer  and  at  its  edges  are  columns  and  groups  of  epi- 
thelial cells,  which,  in  a  single  section,  do  not  join  directly 
with  each  other  or  with  the  overlying  epithelium.  The  cells 
composing  the  columns  are  of  the  same  nature  as  the  prickle 
cells  which  occupy  the  central  rows  of  the  normal  Malpighian 


406  CLINICAL  PATHOLOGY. 

layer  of  the  skin.  They  are  flat,  faintly- staining  cells  with  a 
central  and  usually  lightly-stained  nucleus,  and  their  prickle 
processes  can  often  be  made  out.  Lying  in  the  centre  of  some 
of  the  columns,  or  lying  free  in  the  connective  tissue,  are  found 
in  the  great  majority  of  sections  the  so-called  "cell  nests." 
These  are  round  bodies,  easily  recognised  under  the  f-inch 
objective,  staining  a  bright  red  with  eosin,  and  more  or  less 
structureless.  In  some  cell  nests  the  outlines,  and  more  often 
the  nuclei,  of  the  compressed  elongated  and  curved  epithelial 
cells  can  still  be  made  out.  The  cell  columns  and  nests  lie  in 
a  connective  tissue  stroma,  and,  penetrating  beneath  the  sub- 
epidermal alveolar  tissue,  pass  between  the  muscle  bundles. 
An  inflammatory  exudate  of  cells  is  usually  present  in  the 
stroma  at  the  edges  and  base  of  the  growth. 

A  carcinoma  is  distinguished  from  a  papilloma  by  the 
direction  of  the  growth,  which  is  inwards  and  not  outwards,  by 
the  character  of  the  ramifying  columns  of  epithelial  cells, 
which  involve  the  deeper  structures,  and  by  the  presence  of  cell 
nests. 

The  points  of  distinction  between  a  squamous-celled  carci- 
noma and  a  rodent  ulcer  will  be  given  under  the  description 
of  the  latter  tumour. 

Rodent  ulcer. — Eodent  ulcer  is  an  epithelial  neoplasm 
which  spreads  and  destroys  the  tissues  as  it  grows.  It  is, 
however,  only  locally  malignant  and  never  disseminates,  thus 
differing  from  all  other  varieties  of  carcinoma. 

Rodent  ulcer  affects  the  skin,  and  particularly  the  skin  of 
the  face.  Its  most  characteristic  situations  are  at  the  outer 
canthus  of  the  eye,  on  the  nose,  the  eyelid,  and  less  frequently 
the  pinna :  multiple  rodent  ulcers  are  not  very  uncommon. 
The  histological  aspect  of  a  rodent  ulcer  is  very  characteristic. 
The  growth  arises  from  the  skin  or  from  one  of  the  skin 
appendages,  such  as  the  sebaceous  glands  or  hair  follicles,  and 
is  evidently  a  growth  of  the  palisade  cells  of  the  Malpighian 
layer. 

If  a  section  of  the  normal  skin  be  examined  under  the  micro- 
scope the  basal  row  of  cells  of  the  Malpighian  layer  is  seen  to 
consist  of  tall,  narrow  cells  with  deeply-staining  nuclei.  The 
appearance  of  these  cells  is  in  very  strong  contrast  to  the 
squamous  cells  of  the  more  superficial  layers.  The  same  layer 
of  palisade  cells  is  present  also  in  the  skin  appendages. 


CARCINOMATA— SARCOMATA,   ETC. 


407 


A  section  of  a  rodent  ulcer  shows  columns  of  epithelial  cells 
passing  beneath  the  epidermis.  The  columns  tend  to  be 
longer  and  somewhat  more  regular  than  those  of  a  squamous- 
celled  carcinoma.     They  are  composed  of  the  narrow  elongated 


Fig.  37. — Rodent  Ulcer.     Drawn  under  |-inch  Objective. 


Fig.  38. —Rodent  Ulcer.     Drawn  under  ^-inch  Objective. 


cells  with  deeply-staining  nuclei.  Few  or  no  prickle  cells  are 
present,  consequently  there  is  no  tendency  to  keratinisation 
and  no  formation  of  cell  nests.  An  inflammatory  exudation 
into   the   stroma  is  common,  as  also   into  the  neighbouring 


408  CLINICAL   PATHOLOGY. 

tissues  of  the  majority  of  malignant  growths,  and  particularly 
those  of  cutaneous  surfaces. 

The  distinction  between  squamous-celled  carcinoma  and 
rodent  ulcer  is  rarely  difficult,  since  the  squamous  growth 
is  composed  of  squamous-celled  columns,  with  at  most  an 
imperfect  edging  of  palisade  cells,  and  of  cell  nests,  while  the 
rodent  ulcer  is  composed  of  smaller  elongated  cells  with  deeply- 
staining  nuclei,  very  few  squamous  cells,  and  no  cell  nests. 

Spheroidal-celled  carcinoma.— Spheroidal  or  cuboidal 
celled  carcinoma  originates  from  spheroidal  glandular 
epithelium.  It  is  the  common  carcinoma  of  the  breast  and 
prostate.  It  occurs  also  in  the  ovary,  testis,  pancreas,  thyroid, 
salivary  glands,  and  stomach.  Very  cellular  growths  of  this 
nature  are  known  as  encephaloid  carcinomata,  as  opposed  to  the 
fibrous,  which  are  called  scirrhous  carcinomata.  A  prognosis 
based  upon  the  cellular  character  of  the  growth  is  very  un- 
certain; as  both  forms  of  growth  invade  the  neighbouring 
tissues  and  disseminate  freely  in  the  lymph  glands.  A  fibrotic 
carcinoma  of  the  breast  may  remain  latent  for  a  number  of 
years,  and  after  removal  of  the  main  growth  dissemination 
may  rapidly  occur.  It  is  not  really  possible  to  say  from  the 
histological  findings  in  any  form  of  carcinoma  whether  the 
growth  will  recur  rapidly  or  not.  Carcinoma  in  elderly 
patients  often  shows  very  little  malignancy  :  carcinoma  in 
young  subjects  usually  terminates  fatally  in  a  short  time.  The 
histological  aspect  of  the  growth  in  the  two  cases  is  identical. 

Under  the  microscope  the  spheroidal-celled  carcinoma  is 
seen  to  consist  of  a  number  of  columns  composed  of  oval  cells 
ramifying  in  a  more  or  less  dense  fibrous  stroma.  The  columns 
consist  of  atypical  glandular  acini,  very  irregular  in  shape  and 
size,  and  with  their  lumina  filled  as  a  rule  by  the  cells.  Small 
groups  of  spheroidal  cells  are  characteristically  present  in 
addition,  irregularly  placed  and  lying  free  in  the  fibrous 
stroma.  Some  of  the  cells  may  show  mitotic  figures,  and  these 
may  be  present  in  any  form  of  malignant  neoplasm,  but  afford 
no  particular  evidence  of  malignancy,  since  they  may  occur 
also  in  simple  growths  and  in  normal  tissue. 

Areas  of  cell  degeneration  are  common  in  these  tumours, 
and  particularly  in  the  encephaloid  variety  of  them.  The 
degeneration  consists  of  necrosis  as  a  rule,  but  colloid  change 
is  not  infrequent. 


CARCINOMATA— SARCOMATA,   ETC. 


409 


The  spheroidal-celled  carcinoma  has  to  be  distinguished  from 
an  adenoma  and  from  a  sarcoma. 

The  acini  of  an  adenoma  are  far  more  regular  in  shape  and 
arrangement  than  those  of  a  carcinoma,  and  are  commonly 
composed  of  one  or  two  layers  of  cells  enclosing  a  well-defined 
lumen.     In  the  majority  of  cases  the  distinction  is  easy. 

The  more  degenerated  portion  of  a  carcinoma,  in  which  the 
alveolar  arrangement  is  broken  up,  may  bear  a  considerable 
resemblance  to  a  sarcoma.  The  diagnosis  is  made  clear  by 
examining  also    the   undegenerated   portions.     The   areas   of 


J 

/v; 


■  ?^SWSUri^ 


'       »  ,         VVR':,       *\\&P)J(  V^JttfilP 


mm- 


£;#«       \Ai  \.    %dl)     Vv/-  jHbi!|  /;fjL 


7%  }'/  h  ?#V;^M U^*^^ 


?S^;; 


Fig.  39. — Spheroidal-Celled  Carcinoma  of  Breast. 
Drawn  under  £-inch  Objective. 

degeneration  are  readily  recognised  and  avoided  in  the  fresh 
specimen  by  their  colour  and  soft  consistence.  If  the  growth 
is  entirely  necrotic  it  may  be  impossible  to  distinguish  its 
nature. 

In  examining  breast  sections  in  particular  it  is  essential  to 
have  an  acquaintance  with  the  histology  of  the  normal  gland. 
Areas  of  glandular  activity  in  a  lactating  breast  have  been 
frequently  mistaken  for  malignant  growths. 

The  secondary  deposits  in  glands  have  the  characters  of 
the  primary  growth,  and  considerable  fibrosis  of  the  gland  is 
often  present  in  addition  to  the  alveolar  columns  of  spheroidal 


410 


CLINICAL   PATHOLOGY. 


cells.  The  secondary  deposits  of  all  tumours,  however,  tend  to 
be  more  irregular  than  the  primary  growth,  and  it  may  not  be 
possible  to  tell  their  exact  nature  in  all  cases. 

The  lymph  glands  draining  a  carcinomatous  area  are 
frequently  enlarged,  but  not  necessarily  infected  by  cells  of  the 
neoplasm.  Such  glands  show  great  increase  in  their  epithelioid 
cells,  and  fortunately  these  cellular  areas  hear  little  or  no 
resemblance  to  areas  of  spheroidal  cells.  The  epithelioid 
elements  have,  however,  been  oiten  mistaken  for  malignant 
deposits.  The  glands  show  areas  in  which  the  lymphoid  cells 
are  very  scanty  or  absent,  and  have  been  replaced  by  the  angular 
epithelioid  cells  of  chronic  inflammation. 

Columnar-celled  carcinoma.— This  variety  of  carcinoma 
arises  from,  and  is  mainly  composed  of,  columnar  epithelial 


_<*.<■  C 


Fig.  40. — Columnar- Celled  Carcinoma  of  Colon.     Drawn 
under  g-inch  Objective. 

cells.  It  is  met  with  throughout  the  alimentary  tract  from 
the  pylorus  to  the  commencement  of  the  anal  canal,  in  the 
body  of  the  uterus  and  in  the  gall  bladder  :  also  in  the  larger 
ducts  of  those  glands  which  have  been  mentioned  as  usually 
affected  by  spheroidal-celled  carcinoma.  Columnar-celled 
carcinomata  arising  in  spheroidal-celled  glands  provided  with 
columnar- celled  ducts  are  known  as  duct  carcinomata. 

Under  the  microscope  are  seen   numbers  of   alveoli  lined 
by  a  single  layer  of  columnar  cells,  and  separated  from  each 


CAECINOMATA— SAECOMATA,   ETC.  411 

other  by  a  delicate  and  scanty  connective  tissue.  In  many 
sections  the  columnar-celled  alveoli  differ  bat  little  from  those 
of  the  normal  epithelium,  except  in  the  usual  absence  of 
a  regular  arrangement  relatively  to  each  other.  The  diagnosis 
of  malignancy  in  such  cases  rests  mainly  upon  the  position  of 
the  alveoli,  for  example,  beneath  the  submucous  coats  and 
between  the  muscle  bundles  of  the  muscular  coats  in  the  case 
of  an  intestinal  carcinoma. 

Frequently,  however,  the  alveoli  are  atypical  and  of  very 
irregular  size,  and  in  rapidly-growing  tumours  may  be  so 
compressed  as  to  resemble  rather  solid  columns  of  more  or 
less  spheroidal  cells,  the  alveolar  arrangement  being  present 
only  at  the  growing  edges.  In  other  cases  there  may  be 
proliferation  of  the  lining  cells  of  the  acini  to  form  papuliferous 
processes  projecting  into  the  lumina,  and  a  few  cells  may  be 
seen  passing  out  from  the  lining  layers  into  the  adjacent 
tissues. 

In  secondary  deposits  the  columnar-celled  acini  may  be 
well  formed,  but  they  are  frequently  most  atypical,  and 
difficult  to  distinguish  from  similar  deposits  of  a  spheroidal- 
celled  growth. 

The  Sarcomata. 

The  sarcomata  are  mesoblastic  tumours,  and,  with  the 
exception  of  myeloid  sarcoma,  are  intensely  malignant.  The 
sarcomata  may  very  rarely  disappear  spontaneously  or  after 
the  injection  of  Coley's  fluid.  Cure  of  a  round  or  spindle- 
celled  sarcoma  by  purely  surgical  means  is  extremely  unusual, 
so  rapidly  do  the  secondary  growths  disseminate. 

The  sarcomata  are  classified  mainly  according  to  the  type 
of  cell  of  which  they  are  composed,  and  we  have  to  consider 
round-celled,  spindle-celled,  and  giant-celled  sarcomata.  The 
round-celled  growths  are  further  subdivided  into  small  and 
large  round-celled  sarcomata.  In  addition  are  recognised 
alveolar  and  melanotic  sarcomata. 

The  sarcomata  are  particularly  liable  to  the  various 
degenerations,  and  the  growths  are  commonly  necrosed  in  part. 
In  some  necrotic  growths  it  may  be  impossible  to  obtain  for 
section  a  portion  of  the  tumour  which  sufficiently  indicates 
its  structure.  Mucoid  and  hyaline  degeneration  may  also 
occur.     Sarcomata,  usually  of  the  spindle-celled  variety,  may 


412  CLINICAL  PATHOLOGY. 

be  associated  with  an  excessive  production  of  fibrous  tissue, 
and  such  growths  are  known  as  fibro-sarcomata.  Other 
growths  may  develop  cartilage,  and  are  called  chondro- 
sarcomata ;  others  again  may  produce  bone,  and  are  called 
osteo-sarcomata.  The  majority  of  the  sarcomata  are  very 
soft  and  cellular  tumours,  but  the  varieties  just  mentioned  are 
tough,  and  the  osteo-sarcomata  may  be  of  stony  hardness. 
The  hard  sarcomata  may  disseminate  with  great  rapidity. 
Small  round-celled  sarcoma. — The  distinction  between 


*<?  *—^4tm  ©  ©  ©  Q  ^^ 

Q®  fl^l*     @        ®       @      £*  <§h>  ^      ©         *        f>  fl       ®     ®^ 

»         •  *#*'^  •  @  ©<©©  #"  ©  ^ 

@  ©.  ©  ©  •  ^@  ©|°®   ©@i$  %*  © 

ft    •  •-  »*  %*t**/  ##*'••$*•  2*    • 


Fig.  41. — Small  Bound- Celled  Sarcoma.     Drawn 
under  J-inch  Objective. 

the  two  varieties  of  round-celled  sarcoma  is  not  very  marked, 
and  cells  of  varying  size  are  common  in  the  two  forms. 

The  round-celled  sarcomata  are  the  most  generalised  of  all 
tumours,  and  may  occur  in  any  part  of  the  body,  among  other 
places  in  the  connective  tissues,  and  particularly  in  the  bones, 
in  the  ovary,  testicles,  pharynx,  and  pancreas.  The  round  and 
spindle  celled  sarcomata  of  bones  are  especially  characteristic, 
and  almost  uniformly  run  a  rapid  and  fatal  course.  For- 
tunately they  are  comparatively  rare  tumours. 

Under  the  microscope  the  small  round-celled  sarcoma  is 
seen  to  consist  of  a  scanty  intercellular  connective  tissue  con- 
taining a  large  number  of  cells,  which    show  no   particular 


CARCINOMATA— SARCOMATA,   ETC.  413 

arrangement  in  respect  to  each  other  or  to  the  tissue  in  which 
they  lie.  The  cells  have  a  round  and  usually  reticular  nucleus, 
and  a  small  but  definite  amount  of  cytoplasm.  An  ordinary 
microscopic  field  bears  a  considerable  resemblance  to  an  area 
of  a  lymphatic  gland,  but  there  is  no  glandular  arrangement 
into  cortex  and  medulla,  and  "  germinal  "  centres  are  absent. 
Also  the  individual  cells  of  a  sarcoma  are  definitely  larger 
than  those  found  in  lymph  tissue,  and  are  more  scattered  and 
more  irregularly  placed.  Thin-walled  blood-vessels  and 
hemorrhagic  areas  are  frequent.  Mitotic  figures  are  usually 
numerous. 

Large  round- celled  sarcoma. — This  variety  of  sarcoma 
is  distinctly  less  common  than  the  smaller-celled  tumour. 
The  cells  have  a  big,  round,  deeply-staining  nucleus  and  a 
large  area  of  cytoplasm.  Even  in  paraffin  sections  the  cells 
are  obviously  large,  and  if  unstained  specimens  teased  out 
in  normal  salt  solution  are  examined  the  cells  are  seen  to  be 
very  large  indeed. 

Spindle-celled  sarcoma. — These  sarcomata  are  met  with 
more  frequently  than  the  round-celled  varieties.  They  may  be 
similarly  divided  into  large  and  small  spindle-celled  growths, 
but  the  relative  size  of  the  cells  is  not  materially  important. 
The  tumours  are  met  with  in  much  the  same  situations  as  the 
round-celled  sarcomata,  and  in  particular  in  connective 
tissues,  the  periosteum  of  the  long  bones,  and  in  fasciae. 

Under  the  microscope  the  tumour  is  seen  to  consist  of 
elongated  cells  tapering  to  a  point  at  each  end  and  containing 
a  central  elongated  nucleus.  The  arrangement  of  the  cells 
may  be  fairly  regular,  and  whorls  of  cells  similar  to  but  less 
regular  than  those  met  with  in  fibromata  are  occasionally 
present.  Such  tumours  are  not  often  seen,  are  slowly  grow- 
ing, and  tend  to  recur  locally  rather  than  to  disseminate. 
Occasionally  tumours  are  met  with  in  which  the  spindles  are 
shorter  and  broader,  and  the  growth  is  called  an  "  oat-celled  " 
sarcoma.  In  the  more  typical  sarcoma  the  long  well-formed 
spindle  cells  are  scanty,  and  the  nature  of  the  sarcoma  is  more 
obviously  shown  by  the  spindle-shaped  nuclei  than  by  the  shape 
of  the  cells.  The  arrangement  of  the  cells  is  irregular,  and 
there  are  practically  no  whorls.  Often  the  cells  are  more  or 
less  angular  and  may  show  bifid  processes.  In  nearly  all 
spindle-celled   sarcomata   numerous   round  cells  with  round 


414 


CLINICAL   PATHOLOGY. 


nuclei  are  seen,  and  both  large  and  small  spindle  cells  are 
present.  Such  tumours  are  frequently  called  mixed-celled 
sarcomata.  It  must  be  recognised,  however,  that  the  cells  do 
not  all  lie  in  the  same  direction  and  that  many  are  cut  trans- 
versely and  appear  as  round  or  oval  cells.  In  this  variety  of 
sarcoma,  as  in  other  varieties,  the  great  majority  of  the  blood- 
vessels are  mere  spaces  in  the  reticulum,  and  their  walls  are 
rarely  composed  of  more  than  one  layer  of  endothelial  cells. 


*&r  U£  'AnKi  '-'WAV 


»»  Q 


„  9  t 


•a 


*  -%  ,  1 W  rf  6o  viWfi  eUM  »#W 


l_a_ 


Pig.  42. — Spindle-Celled  Sarcoma.     Drawn 
under  jl-inch  Objective. 

The  character  of  the  blood-vessels  in  a  sarcoma   is  of  the 
utmost  diagnostic  importance. 

Myeloid  sarcoma.— It  is  doubtful  whether  this  tumour 
should  be  classed  as  a  sarcoma.  It  is  an  endosteal  tumour,  which 
invades  and  destroys  the  surrounding  tissues,  but  scarcely  ever 
forms  secondary  deposits.  Local  removal,  and  often  the 
necessarily  incomplete  local  removal  by  curettage,  is  usually 
sufficient  for  a  cure.  The  growth  is  called  by  some  a  myeloma 
in  preference  to  a  myeloid  or  giant-celled  sarcoma.  Myeloid 
sarcoma  is  common  in  the  maxilla,  forming  one  variety  of 
epulis,  and  is  frequently  met  with  in  the  extremities  of  the 
long  bones,  particularly  in  the  upper  end  of  the  tibia  and  in 
the  lower  end  of  the  femur. 


CARCINOMATA— SARCOMATA,   ETC.  415 

Under  the  microscope  the  character  of  the  growth  is 
extremely  typical.  The  essential  feature  is  the  presence  of 
abundant  myeloid  or  giant  cells.  These  cells  are  large,  and 
both  variable  and  irregular  in  size  and  shape,  the  majority 
being  more  or  less  oval.  They  contain  very  many  nuclei, 
often  20  or  more,  scattered  irregularly  throughout  the 
cytoplasm.  Giant  cells  of  a  somewhat  similar  type  may  be 
present  in  ordinary  granulation  tissue,  and  exactly  similar 
cells  are  frequently  found  in  an  inflammatory  process  con- 
nected with  bone.     In  no  other  tissue,  however,  are   to   be 


Fig.  4.'3. — Myeloid  Sarcoma.     Drawn  under  f-incli  Objective. 

found  so  many  of  such  cells  as  to  form  the  predominant 
feature  of  a  microscopic  field. 

In  addition  to  the  giant  cells  is  found  tissue  precisely 
similar  to  that  which  mainly  comprises  a  spindle-celled 
sarcoma  of  the  small  irregular  spindle-celled  type.  The 
spindle-celled  parts  of  the  tumour  penetrate  among  the  myeloid 
cells,  and  also  form  areas  free  from  giant  cells.  The  blood- 
vessels are  very  numerous  and  thin-walled,  and  hemorrhages 
into  the  growth  are  frequent.  The  hemorrhagic  character 
of  the  growth  gives  to  the  fresh  specimen  a  characteristic 
maroon  colour. 

Alveolar  sarcoma. — This  name  is  given  to  sarcomata  in 
which   the  cells  have  an  irregular  arrangement  into  alveoli 


416  CLINICAL   PATHOLOGY. 

separated  from  each  other  by  strands  of  fibrous  tissue.  The 
alveolar  sarcomata  are  most  commonly  seen  in  connection 
with  the  skin,  and  may  arise  from  moles.  They  are  often 
melanotic.  The  cells  in  an  alveolar  sarcoma  are  practically 
always  of  the  small  angular  spindle-shaped  variety. 

Melanotic  sarcoma. — This  tumour  is  confined  to  situations 
in  which  melanin  is  normally  present  :  consequently  it  is 
found  in  the  skin,  and  particularly  in  pigmented  moles,  and  in 
the  choroid.  It  less  frequently  arises  in  the  nail  matrix  of 
the  fingers,  or  in  the  adjoining  skin.     Melanotic  sarcomata 


:**- 


of 

^      ^>     ^  G— 


Fig.  44. — Melanotic  Alveolar  Sarcoma.     Drawn 
under  ^-inch  Objective. 

developing  in  moles  always  show  evidence  of  an  alveolar 
arrangement.  Such  moles  may  remain  quiescent  for  years, 
suddenly  become  malignant,  and  disseminate  with  great 
rapidity. 

The  amount  of  pigment  is  variable,  and  it  is  usual  for  the 
secondary  growths  to  be  more  deeply  pigmented  than  the 
primary  tumours. 

The  secondary  deposits  occur  in  the  neighbouring  lymph 
glands,  and  from  thence  disseminate  to  the  liver,  lung,  kidney, 
brain,  and  other  organs. 

The  histological  diagnosis  of  a  secondary  deposit  is  simple : 
that  of  the  primary  growth  in  the  skin  is  often  difficult,  and 


CABCINOMATA— SARCOMATA,   ETC.  417 

has  been  referred  to  in  the  paragraph  dealing  with  pigmented 
moles. 

Othee  Tumoues. 

Other  malignant  growths,  difficult  to  classify  or  not 
included  in  the  above  account,  are  referred  to  here.  They 
include  deciduoma  malignum,  renal  tumours,  and  teratomata. 

Deciduoma  malignum. — Deciduoma  malignum,  or  chorion 
epithelioma,  is  a  rare  malignant  growth  of  the  uterus  which 
has  been  variously  classified  as  a  sarcoma  and  a  carcinoma. 
The  growth  follows  a  pregnancy,  and  in  a  large  number  of 
cases  has  been  associated  with  a  hydatidiform  mole.  It  is  an 
extremely  malignant  tumour,  and  rapidly  disseminates  to  the 
lungs  and  other  parts  of  the  body.  Very  similar  tumours 
may  rarely  occur  in  the  testicle. 

The  growth  is  very  vascular,  and  haemorrhages  into  it  are 
the  rule. 

Under  the  microscope  the  growth  is  seen  to  consist  as  a  rule 
mainly  of  blood  clot  enclosing  comparatively  few  and  small 
cellular  processes.  Some  of  the  processes  may  show  a  tubular 
arrangement,  and  in  the  majority  of  them  are  to  be  found  cells 
of  two  varieties  representing  the  two  layers  of  a  chorionic 
villas.  The  smaller  cells  from  the  investing,  or  syncytial,  layer 
are  seen,  together  with  the  large  polyhedral  cells  known  as 
decidual  cells,  and  derived  from  the  inner  or  Langhan's  layer. 

Renal  tumours. — The  kidney  may  be  affected  by  a  variety 
of  new  growths  common  to  other  tissues,  but  all  are  rare. 
One  remarkable  tumour  must  be  mentioned  here,  and  is 
apparently  peculiar  to  the  kidney.  The  tumour  is  known  as 
a  hypernephroma,  or  as  a  von  Grawitz  tumour.  It  is  a 
malignant  tumour,  probably  of  carcinomatous  nature,  and 
is  supposed  to  originate  in  adrenal  gland  inclusions.  The 
tumour  may  occur  in  young  children  or  in  middle  life.  Com- 
mencing in  the  upper  pole  of  the  kidney  it  gradually  extends 
downwards,  compressing  the  renal  substance,  and  remaining 
for  a  considerable  period  more  or  less  encapsulated.  It  finally 
burrows  its  way  down  to  the  renal  pelvis  and  grows  into  the 
vessels,  whence  it  disseminates  over  the  body. 

Under  the  microscope  the  tumour  is  very  characteristic. 
Areas  of  haemorrhage  and  degeneration  are  common,  but  in 
the   undamaged    growth    areas   is    seen    a   capillary-bearing 

p.  '27 


418  CLINICAL  PATHOLOGY. 

stroma  enclosing  more  or  less  circular  collections  of  cells. 
The  cells  are  swollen,  round  or  polygonal,  contain  a  central 
nucleus,  and  often  stain  somewhat  poorly.  The  cells  may  be 
arranged  in  columns,  the  centres  of  which  are  often  degenerated 
and  thus  convey  the  impression  of  alveolar  processes. 

The  tumours  contain  a  large  amount  of  glycogen  and  fat, 
and  were  originally  described  by  von  Grawitz  as  fatty  tumours 
of  the  kidney. 

Teratomata. — These  tumours  are  new  growths  which 
contain  a  variety  of  tissue  elements.  The  teratomata  include 
the  dermoid  cysts  of  the  ovary,  in  which  any  of  the  skin 
appendages,  such  as  hair,  nails  and  teeth,  may  be  found,  as 
well  as  other  cysts,  which  may  contain  fragments  of  numerous 
internal  organs.  Such  teratomata  may  occur  in  the  testis  as 
well  as  in  the  ovary.  The  malignant  teratomata  are  remark- 
able mixed  tumours  which  may  contain  sarcomatous  and 
carcinomatous  tissue.  The  former  are  often  spindle  celled, 
and  the  latter  columnar.  The  sarcomatous  and  epithelial 
growths  may  appear  side  by  side  in  the  same  section.  Areas 
of  well-formed  cartilage  are  common  in  such  tumours,  while 
muscle  fibres  and  almost  any  variety  of  tissue  may  be  present 
in  addition. 

The  teratomata  have  been  considered  to  represent  abortive 
impregnations.  It  has  been  suggested  that  the  ova  of  the 
embryo  have  been  fertilised  by  the  father  of  the  embryo,  and 
that  the  teratoma  is  the  imperfect  individual  resulting  from 
this  achievement. 

Cysts. 

Cysts  are  pathological  formations  enclosed  by  a  membrane, 
and  containing  fluid  or  semi-fluid  material.  The  majority 
of  cysts  result  from  the  dilatation  of  secreting  tubules 
following  obstruction  to  the  outflow  of  the  secretion.  The 
tubule  may  be  normal,  and  the  obstruction  the  result  of 
acquired  pathological  change  outside  the  secreting  tissue,  as  in 
the  retention  cysts  of  the  kidney  or  breast ;  or  the  cyst  may 
arise  in  a  persistent  rudimentary  remnant  of  foetal  tissue,  as  in 
the  case  of  a  branchial  or  parovarian  cyst.  All  such  cysts  are 
known  as  retention  cysts. 

Other  cysts  may  arise  as  the  result  of  softening  processes  in 


CARCINOMATA— SAECOMATA,  ETC.  419 

solid  tissues,  or  may  be  due  to  the  presence  of  parasites.     Only 
the  more  important  cysts  can  be  mentioned  here. 

Retention  cysts. — Retention  cysts  result  from  the  blocking, 
and  most  commonly  from  the  incomplete. blocking,  of  the  duct 
of  a  secreting  gland.  One  of  the  best  known  examples  is  the 
ranula,  a  cyst  arising  in  connection  with  the  submaxillary 
gland.  Pancreatic  cysts  may  reach  a  considerable  size,  as 
also  may  the  single  cysts  of  the  parovarium.  The  majority 
of  such  retention  cysts  are  of  little  histological  interest,  since 
the  lining  membrane  is  usually  composed  of  more  or  less 
structureless  fibrous  tissue.  The  sebaceous  cyst,  however, 
has  a  well-developed  epithelial  lining,  and  some  of  the  cysts  of 
vestigial  structures  are  of  histological  importance.  The 
dermoid  cyst,  resulting  from  an  epidermal  inclusion,  has  a 
lining  membrane  which  contains  all  the  layers  of  the  true  skin, 
including  the  stratum  granulosum.  The  branchial  cyst 
of  the  neck  has  an  epithelial  lining,  often  composed  of  columnar, 
and  sometimes  of  columnar  ciliated,  epithelium. 

Cysts  of  a  similar  nature  may  arise  in  the  substance  of 
a  secreting  organ,  and  may  follow  fibrosis  of  the  organ 
resulting  from  toxic  or  inflammatory  processes,  the  fibrosis 
constricting  the  duct  lumina.  Such  cysts  are  common  in  the 
kidney  and  the  breast. 

Cysts  may  also  develop  in  the  adenomata  of  secreting  glands, 
and  are  commonly  found  in  the  breast  and  in  the  thyroid. 
Such  cystic  formations  are  known  as  cystic  adenomata.  The 
cysts  in  such  cases  are  due  to  retention  of  secretion  in  the 
abnormal  alveoli,  and  do  not  always  contain  the  normal 
secretion  of  the  organ.  They  are  lined  with  a  definite 
epithelium,  usually  more  or  less  columnar.  The  multilocular 
ovarian  cyst  is  of  this  nature,  and  is  known  as  a  "  cystoma." 
The  epithelial  cyst  lining  of  the  ovarian  cystoma  is  often 
composed  of  flattened  and  not  of  columnar  cells,  the  flattening 
of  the  cells  being  ascribed  to  the  pressure  of  the  fluid  in  the 
cysts. 

In  fibro-cystic  disease  of  the  breast  the  sections  may  show 
considerable  areas  of  fibrosis  with  little  cellular  evidence  of 
past  inflammation,  and  numerous  cysts  of  very  variable  size. 

Congenital  cystic  disease  of  the  kidney  is  almost  invariably 
bilateral,  and  nearly  the  entire  area  of  both  kidneys  may  be 
converted  into  a  number  of  thin-walled  cysts. 

27-2 


420  CLINICAL   PATHOLOGY. 

Degeneration  cysts. — These  cysts  are  not  of  great 
importance.  They  are  not  infrequent  in  new  growths  as  the 
result  of  necrosis  and  softening  of  portions  of  the  growth,  and 
in  old  haemorrhages.  Such  collections  of  fluid  are  scarcely 
true  cysts  in  that  they  have  no  true  limiting  membrane. 

Parasitic  cysts. — The  occurrence  of  these  cysts  has  been 
noticed  in  the  description  of  the  higher  parasites. 


CHAPTER  XXIX. 


HISTOLOGICAL    METHODS. 


The  tissues  removed  at  operation  are  preferably  received 
into  the  laboratory  without  the  addition  of  any  preserving 
fluid.  The  constant  handling  of  tumours  gives  the  pathologist 
a  considerable  acquaintance  with  their  various  appearances, 
and  the  naked-eye  observation  is  of  great  assistance  to  him. 
The  addition  of  alcohol  or  formalin  to  the  tissues  completely 
alters  their  appearance,  and  tends  to  obscure  the  relationship 
of  the  parts  removed.  If,  for  geographical  reasons,  a 
preservative  is  necessary,  the  tissues  may  be  placed  in 
50  per  cent,  methylated  spirit  or  in  10  per  cent,  formalin. 

It  is  also  advisable  that  as  much  of  the  material  should  be 
obtained  as  possible  in  order  that  the  relationship  of  the  normal 
and  abnormal  tissues  can  be  noted,  and  portions  removed  from 
the  most  desirable  places. 

The  selection  of  the  portion  or  portions  to  be  sectioned 
must  be  made  with  considerable  care.  It  is  a  common  error  to 
remove  pieces  of  far  greater  surface  area  and  thickness  than 
are  required  for  diagnostic  purposes.  In  cases  of  evident 
tumour  the  firmest  and  least  necrotic  portion  should  be  chosen 
from  the  growth  itself,  and  in  the  majority  of  cases  a  second 
portion  at  the  junction  of  growth  and  normal  tissue.  If  more 
than  one  variety  of  tissue  is  present,  portions  should  be 
selected  from  each  variety.  Metastatic  growths  in  lymph 
glands,  or  elsewhere  from  the  same  case,  should  also  be 
examined.  Lymph  glands,  even  if  normal  to  the  naked  eye, 
should  always  be  sectioned.  Small  glands  lying  in  a  mass  of 
fat  are  most  readily  recognised  by  their  firm  and  shotty 
consistence. 

The  direction  in  which  the  section  is  to  be  cut  is  frequently 
of  great  importance,  particularly  in  the  case  of  tumours 
or  ulcers  of  the  skin  and  of  the  intestinal  tract.  It  is  often 
advisable  to  make  a  rough  sketch  of  the  portion  removed 
for  examination,  with  an  arrow  indicating  the  surface  to  be  cut. 


422  CLINIC  AX   PATHOLOGY. 

Minute  fragments  of  friable  tissue  which  have  been  removed 
by  the  curette  are  best  placed  in  a  layer  of  gauze  tied  with  a 
thread  in  the  form  of  a  small  sac.  The  tissues  can  remain 
in  the  gauze  sac  until  the  stage  of  "  casting  "  in  the  paraffin 
process. 

Tissues  containing  bone  must  be  decalcified  by  a  special 
process,  and  it  is  well,  if  possible,  to  examine  separately  one 
portion,  free  of  bone,  by  the  ordinary  method. 

Tissues  to  be  examined  for  the  presence  and  distribution  of 
fat  must  pass  through  the  gum,  and  not  the  paraffin,  process, 
since  the  latter  involves  passage  through  alcohol. 

The  methods  of  fixing,  cutting,  and  staining  sections  are 
very  numerous.  Only  those  are  given  here  which  are  generally 
suitable  for  the  diagnosis  of  the  great  majority  of  tissues 
received  from  the  wards  and  operating  theatres.  The  processes 
of  fixing  and  cutting  tissues  described  here  are  those  of  gum  and 
paraffin.  The  preparation  of  celloidin  sections  is  rarely  called 
for  in  clinical  pathology,  and  is  practically  only  required  for 
sections  through  the  globe  of  the  eye. 

The  preparation  of  gum  sections.— The  gum  method 
has  numerous  advantages,  and,  since  it  requires  the  minimum 
of  apparatus,  is  to  be  recommended  for  diagnostic  purposes. 
Bather  more  practice  is  required  for  the  actual  cutting  of  the 
section,  unless  one  of  the  more  elaborate  section  cutters,  such 
as  the  excellent  one  of  Delepine,  is  available. 

The  apparatus  required  consists  of  a  hand  microtome  and  a 
gum-embedding  solution. 

The  gum  solution  is  made  as  follows  : — 

Saturate  gum  acacia  in  hot  water. 

Mix  3  parts  of  the  gum  solution  with  1  part  of 
syrup  (B.  P.). 

The  most  convenient  pattern  of  microtome  is  Williams' 
microtome,  made  by  Messrs.  Swift  &  Son.  It  consists  of  a 
circular  plate  fitted  with  a  clamp,  which  can  be  attached  to 
the  edge  of  a  firm  table,  and  of  an  ether  spray  apparatus, 
by  means  of  which  a  spray  of  ether  can  be  directed  against 
the  under  surface  of  a  small  metal  disc  let  into  the  circular 
plate.  An  ordinary  razor  blade  is  fitted  into  a  brass  tripod 
carrier  at  a  fixed  angle.  The  level  of  the  cutting  edge  is 
regulated  by  a  screw  at  the  apex  of  the  tripod. 

An  ethyl-chloride  spray  is  an  extremely  useful  adjunct. 


HISTOLOGICAL   METHODS. 


423 


There  are  two  methods  of  conducting  the  process,  a  rapid 
and  a  slow  one. 

The  rapid  method.— The  rapid  method  can  be  performed 
with  the  majority  of  tissues,  and  is  particularly  useful  for  the 
immediate  diagnosis  of  tumours  during  an  operation.     The 


Fig.  45. — Williams'  Freezing  Microtome. 


method  requires  some  practice,  and  the  interpretation  of  the 
results  must  in  most  cases  be  left  to  the  expert,  since  the 
appearance  of  rapidly-frozen  gum  sections  is  very  different 
from  that  of  the  more  familiar  paraffin  section.  In  many 
instances  the  tissue  can  be  cut,  stained,  and  reported  on  within 
10   minutes    of    removal   from   the   body.      The   method   is 


424  CLINICAL   PATHOLOGY. 

therefore  very  useful,  but  in  a  proportion  of  cases  sections  of 
tumours  requiring  long  and  careful  examination  are  met  with, 
and  a  certain  diagnosis  cannot  and  should  not  be  made  on 
the  sj)ot.  The  diagnosis,  unless  extremely  obvious,  should 
always  be  confirmed  by  sections  made  at  leisure. 

Eapid  sections  have  the  great  advantage  that  practically  no 
shrinkage  of  the  tissues  takes  place,  and  they  are  for  this 
reason  to  be  preferred  to  the  paraffin  section.  On  the  other 
hand,  the  clearing,  staining,  and  flattening  out  of  the  section 
are  less  satisfactory  in  the  majority  of  cases. 

The  process  is  as  follows  : — 

With  a  sharp  scalpel  remove  the  most  suitable  portion  of 
the  tissue. 

Place  the  portion  on  the  central  disc  of  the  microtome  stand. 

Just  cover  it  with  gum  solution.  Do  not  pour  on  so  much 
gum  solution  that  it  flows  over  the  edge  of  the  disc.  Freeze 
with  the  ether  spray  from  below,  assisted  by  the  ethyl-chloride 
spray  from  above. 

The  freezing  is  complete  when  the  gum  is  quite  white  and 
the  tissue  firm  to  the  touch.  If  the  freezing  process  is  con- 
tinued too  long  the  tissue  becomes  extremely  hard  and  cannot 
be  cut  at  all,  in  which  case  thawing  may  be  hastened  by  moisten- 
ing tissue  and  gum  with  warm  water.  If  the  freezing  is 
insufficient  the  gum  is  readily  dented  with  slight  pressure  of 
the  finger,  and  the  tissue  leaves  its  bed  when  the  razor  meets  it. 

The  freezing  process  should  be  completed  in  a  few  minutes. 

Moisten  the  stage  of  the  microtome  with  water  to  allow  the 
razor  carrier  to  slide  easily. 

Adjust  the  level  of  the  razor  edge  exactly  to  the  height  of 
the  tissue. 

Hold  the  razor  carrier  in  both  hands,  with  the  forefinger  of 
the  right  hand  resting  on  the  front  adjusting  screw. 

Eapidly  sweep  the  razor  across  the  tissue,  keeping  the 
carrier  legs  pressed  against  the  microtome-stand  surface. 

With  the  forefinger  turn  the  screw  a  short  distance  onwards 
to  depress  the  cutting  edge.  The  amount  of  the  turn  deter- 
mines the  thickness  of  the  section  cut. 

Eepeat  the  process  about  a  dozen  times  in  rapid  succession. " 

With  a  small  camel-hair  brush  wipe  gently  the  mixture  of 
gum  and  tissue  from  the  upper  surface  of  the  razor  blade  into  a 
tall  glass  beaker  filled  with  warm  normal  saline. 


HISTOLOGICAL   METHODS.  425 

The  sections  float  out  on  the  surface  of  the  fluid,  and  can  be 
assisted  to  separate  by  gently  touching  with  the  brush. 

If  complete  and  thin  sections  do  not  appear,  cut  more,  either 
of  different  thickness  or  after  altering  the  consistence  of  the 
gum  and  tissue  by  further  freezing  or  thawing. 

Pick  up  the  best  section  on  a  clean  glass  slide  by  holding 
the  slide  vertical  and  submerged  in  the  saline  and  drawing  it 
upwards  along  the  section.  If  the  section  is  curled  it  may 
often  be  straightened  out  by  partially  floating  it  off  again  in 
the  saline  with  the  aid  of  a  blunt  mounted  needle  or  probe. 

Drain  off  the  fluid  as  much  as  possible. 

Drop  on  the  section  1  drop  of  Loffler's  methylene  blue. 

Carefully  let  down  a  cover-slijD  in  the  slain  over  the  section. 

Press  the  cover-slip  down  with  2  mounted  needles. 

Wipe  off  the  excess  of  stain  from  around  the  cover-slip. 

Examine  with  a  §-inch  and  ^-inch  objective. 

A  differential  stain,  and  a  more  permanent  preparation,  can 
be  made  by  the  rapid  method  as  follows  : — 

Take  up  the  section  from  the  salt  solution  on  a  section  lifter 
or  a  blunt  needle,  and  leave  it  in  hsemalum  for  2  minutes. 

Transfer  to  tap  water  in  a  tall  beaker  for  2  minutes. 

Pick  up  on  a  slide,  and  flatten  out  by  smoothing  with  a 
cigarette  paper  moistened  in  water.  Peel  off  the  cigarette 
paper  carefully. 

Cover  slide  and  section  with  5  per  cent,  alcoholic  eosin  for 
1  minute. 

Cover  with  methylated  spirit  for  a  few  seconds. 

Cover  with  absolute  alcohol  for  1  minute. 

Cover  with  xylol  for  1  minute. 

Drain  off  xylol,  and  mount  in  Canada  balsam. 

The  slow  method. — By  this  method  the  tissues  are  first 
fixed,  then  cut  and  stained  at  leisure.  The  method  is  par- 
ticularly useful  for  the  demonstration  of  fat  in  tissues. 

The  method  of  staining  fat  in  tissues. 

Place  the  tissues  selected  in  salt  formalin  solution  of  the 
following  composition  :— 

Water 100  c.c. 

Commercial  formalin  (i.e.,  40  per  cent.)   .     10    ,, 
Sodium  chloride      .....       1  gramme. 

Leave  in  salt  formalin  for  24  hours. 

Wash  in  running  water  for  24  hours. 


426  CLINICAL   PATHOLOGY. 

Transfer  to  gum  solution  (see  above)  for  24  hours. 

Cut  sections  with  freezing  microtome. 

Place  sections  in  water  for  1  hour. 

Transfer  to  a  small  well-stoppered  bottle  filled  with 
Scharlach  R.  for  36  hours. 

Transfer  to  75  per  cent,  spirit  for  a  minute  or  two. 

Transfer  to  water.  The  sections  spin  round  rapidly  and 
spread  out  on  the  surface  of  the  water. 

Place  in  filtered  hsBmalum  for  1  minute. 

Transfer  to  tap  water  in  a  tall  beaker  for  3  minutes. 

Pick  up  on  a  slide.  Drain  off  the  excess  of  water,  and 
mount  in  Farrant's  solution. 

The  fat  is  stained  red  and  the  tissues  blue. 

If  the  demonstration  of  fat  is  not  required  proceed  as 
follows  : — 

Fix  in  the  same  manner,  and  after  cutting  the  sections  leave 
in  water  for  about  an  hour. 

Then  stain  with  hsemalum  and  eosin  in  the  manner  described 
for  the  rapid  method,  varying  the  time  of  staining  according 
to  the  nature  of  the  tissue,  and  mount  in  Canada  balsam. 

The  preparation  of  paraffin  sections. 

1.  The  fixation  of  the  tissues.— This  can  be  done  by  one 
of  the  two  following  methods.  The  alcohol  method  is  quite 
reliable  for  the  majority  of  tissues,  and  furnishes  very  suitable 
sections  for  diagnostic  purposes.  Fixation  by  Zenker's  fluid 
is  suitable  for  small  pieces  of  tissue,  and  particularly  for  soft, 
friable  growths.  This  method  gives  better  fixation  than  the 
simple  alcohol  process,  and  should  be  used  when  exact  cellular 
details  are  required. 

A.  The  alcohol  method. — Place  the  tissue  selected  in 
50  per  cent,  alcohol  for  24  hours. 

Transfer  to  90  per  cent,  alcohol  or  methylated  spirit  for 
24  hours. 

Transfer  to  absolute  alcohol  for  4  hours. 

Transfer  to  xylol.  If  the  xylol  becomes  cloudy  return  to 
absolute  alcohol  for  2  more  hours  in  order  to  get  complete 
dehydration. 

Leave  in  xylol  from  12  to  24  hours,  or  until  the  tissue 
becomes  transparent. 

Remove  from  xylol,  and  blot  off  the  excess  of  xylol  with 
blotting  paper. 


HISTOLOGICAL   METHODS.  427 

Place  in  melted  paraffin  in  an  incubator  kept  at  125°  F. 
The  paraffin  should  have  a  melting  point  of  about  120°  F. 
and  should  not  be  heated  to  a  higher  temperature  than  is 
necessary  to  keep  it  in  a  liquid  state. 

After  from  8  to  12  hours,  during  which  the  paraffin  is  changed 
three  times,  there  should  be  no  smell  of  xylol  detected  in 
the  paraffin. 

The  tissue  is  then  ready  to  "  cast." 

Two  L  embedding  blocks  are  arranged  to  form  a  square  on 
any  smooth,  hard  surface,  such  as  a  piece  of  glass,  which  has 
previously  been  lightly  smeared  with  vaseline.  The  blocks 
should  also  have  their  inner  surfaces  smeared  with  vaseline. 
The  tissue  is  removed  from  the  paraffin  bath  with  a  pair  of 
forceps,  previously  warmed  at  the  tips  in  a  gas  flame.  The 
square  is  then  filled  with  melted  paraffin  and  the  tissue  care- 
fully laid  in  it  so  that  the  surface  to  be  cut  is  lying  flat  and 
facing  directly  downwards  against  the  glass.  When  the 
paraffin  block  has  set  hard  the  L  blocks  are  knocked  away,  and 
the  paraffin  block  is  placed  in  a  labelled  chip  box  until  required 
for  sections. 

The  entire  process  takes  from  5  days  to  a  week,  but  may  be 
considerably  hastened  if  desired.  A  paraffin  section  can  if 
necessary  be  completed  in  one  day. 

To  hasten  the  process  it  is  essential  to  select  small  pieces 
of  tissue  for  examination.  The  surface  area  of  the  portion 
removed  is  less  important  than  its  thickness,  which  should 
be  reduced  as  much  as  possible  with  a  sharp  knife.  The 
tissue  is  placed  in  methylated  spirit  for  about  1  hour,  then 
in  absolute  alcohol  for  2  hours,  then  in  xylol  till  clear  ;  then 
in  liquid  paraffin,  which  must  be  changed  every  half  honr 
until  the  smell  of  xylol  is  no  longer  detected,  when  the  block 
is  cast.  Very  excellent  sections  can  often  be  obtained  in 
this  manner  provided  a  very  thin  portion  of  tissue  is  carried 
through  the  fluids. 

The  various  strengths  of  alcohol  and  the  xylol  can 
be  used  for  several  specimens.  Each  specimen  must  be 
kept  in  a  separate  small  bottle  securely  corked,  and 
the  bottle  must  be  emptied  out  thoroughly  before  the 
next  fluid  is  added.  The  melted  paraffin  can  be  kept  in 
small  glass  pots  without  a  cork  ;  and  a  well-regulated  oven, 
or   incubator   kept   at    the   appropriate   temperature,    is   an 


428  CLINICAL   PATHOLOGY. 

advantage.  The  oven  can  be  dispensed  with  and  the  paraffin 
pots  can  be  kept  in  a  water  bath  the  temperature  of  which  is 
regulated  by  the  size  of  the  flame  under  it.  Such  an  appara- 
tus must  be  periodically  inspected  in  order  to  be  sure  that 
the  paraffin  remains  liquid  and  the  temperature  does  not  rise 
too  high. 

B.  Fixation  in   Zenker's   fluid. — This   fluid    has    the 
following  composition : — 

Potassium  bichromate       .         .         .     2'5  grammes. 


Sodium  sulphate 
Corrosive  sublimate 
Glacial  acetic  acid 
Water  to    . 


1  gramme. 

5  grammes. 

.     5  c.c. 

100  „ 


The  corrosive  sublimate  and  the  potassium  bichromate  are 
dissolved  in  the  water  by  heating. 

If  a  considerable  amount  of  stock  solution  is  made  up  it  is 
advisable  to  omit  the  acetic  acid,  adding  it  in  the  proper 
proportion  as  required. 

The  tissue  to  be  fixed  should  be  cut  moderately  thin  and 
placed  direct  in  the  Zenker's  fluid  and  left  for  from  8  to  12 
hours.  The  tissue  is  then  washed  in  running  water  for 
24  hours;  then  placed  in  30  per  cent,  spirit  for  about  4  hours, 
then  in  60  per  cent,  spirit  for  the  same  time,  then  in 
methylated  spirit  containing  sufficient  tincture  of  iodine  to 
give  it  a  pale  port  wine  colour,  then  in  absolute  alcohol,  with 
two  changes,  for  about  4  hours.  The  remainder  of  the  process 
is  the  same  as  in  the  alcohol  fixation  method. 

The  object  of  the  tincture  of  iodine  in  the  methylated  spirit 
is  to  dissolve  out  of  the  tissue  the  mercury  which  tends  to  pre- 
cipitate from  the  Zenker's  fluid.  The  period  in  absolute  alcohol 
and  the  change  of  alcohol  must  be  sufficient  to  discharge  the 
iodine  from  the  tissue. 

C.  Decalcification  of  tissues.— If  much  bone  is  present 
in  the  tissue  it  is  necessary  to  decalcify. 

Before  decalcifying  the  tissue  must  be  fixed  and  treated  by 
method  A  or  B  until  the  end  of  the  alcohol  stage. 

The  tissue  is  transferred  from  alcohol  to  the  decalcifying  fluid. 

The  fluid  should  be  made  as  follows  : — 

Add  carefully  in  the  fume  closet  1  gramme  of  phloroglucin 
to  10  c.c.  of  nitric  acid.  Keep  for  at  least  24  hours,  or  until 
the  reddish  mixture  has  become  light  yellow. 


HISTOLOGICAL   METHODS.  429 

Dilute  with  100  c.c.  of  10  per  cent,  nitric  acid  in  water. 

The  extent  of  decalcification  can  be  tested  by  probing 
the  tissue  with  a  needle.  The  process  takes  3  or  4  hours  as 
a  rule  in  the  above  solution,  but  may  take  longer.  After 
decalcification  remove  the  tissue  to  running  water  for  24  hours. 
Return  to  methylated  spirit  for  24  hours,  then  to  absolute 
alcohol,  and  continue  as  in  A  and  B. 

2.  The  cutting-  of  the  sections. — A  special  microtome 
is  required,  and  the  apparatus  most  generally  used  is  the 
Cambridge  rocking  microtome. 

The  procedure  is  as  follows  : — 

Pare  down  the  paraffin  block,  leaving  a  small  margin  of 
wax  round  the  tissue. 

Heat  a  metal  spatula  in  the  flame  and  melt  with  the  hot 
spatula  the  base  of  the  paraffin  block.  Press  down  the  block 
on  to  the  metal  carrier  so  that  the  surface  of  the  tissue  to  be 
cut  is  horizontal. 

Adjust  the  carrier  so  that  the  block  surface  just  touches 
the  razor  blade. 

Begin  by  cutting  the  thickest  possible  sections  until  the 
entire  surface  area  of  the  tissue  is  exposed :  then  adjust  the 
pointer  to  the  required  mark  on  the  scale.  The  majority  of 
tissues  should  be  cut  at  from  5  to  7m  thick. 

In  cutting,  move  the  handle  of  the  microtome  with  a  rapid 
and  even  movement.  Provided  the  razor  is  really  sharp,  very 
little  practice  is  required.  Discard  all  broken  and  imperfect 
sections.  It  is  best  at  first  to  cut  one  section  at  a  time,  and 
not  a  ribbon  of  sections.  Each  section  is  removed  and 
discarded  until  a  perfect  section  is  obtained. 

Difficulty  in  cutting  a  tissue  which  should  be  reasonably 
soft  usually  means  that  the  xylol  was  imperfectly  removed  in 
the  paraffin  bath.  The  xylol  is  readily  detected  in  the  block  by 
its  smell  and  gritty  consistence,  and  the  block  must  be  returned 
to  the  paraffin  bath  and  subsequently  re-cast. 

Remove  from  the  razor  blade  the  thinnest  sections  with  the 
aid  of  a  small  piece  of  paper  and  a  mounted  needle. 

Float  the  sections  selected  in  a  tall  beaker  filled  with  warm 
water. 

The  water  should  feel  distinctly  warm  to  the  touch  and 
should  be  sufficiently  hot  to  spontaneously  flatten  out  any 
wrinkles  in  the  section,  but  not  so  hot  as  to  melt  the  paraffin. 


430  CLINICAL   PATHOLOGY. 

As  soon  as  the  section  is  quite  flat  it  is  picked  up  by  drawing  a 
clean  slide,  which  has  been  thoroughly  rinsed  in  hot  running 
water,  along  the  section,  in  such  a  way  that  the  section  clings 
to  the  centre  of  the  slide.  The  slide  is  held  vertically  with 
the  majority  of  its  length  in  the  water  and  drawn  out  slowly 
in  a  vertical  direction  when  the  section  floats  against  it. 
The  excess  of  water  is  wiped  off  the  slide.  A  strip  of  stout 
clean  paper  is  soaked  in  methylated  spirit  and  laid  along  the 
slide  over  the  section,  which  is  then  firmly  smoothed  down 
with  the  forefinger  moistened  in  spirit.  The  paper  is  then 
peeled  off  and  the  slide  wiped. 

After  the  sections  are  mounted  the  slides  are  put  in  a  warm 
place,  such  as  an  incubator  at  37°  C,  until  perfectly  dry.  They 
are  then  ready  to  be  cleared  and  stained,  or  can  be  kept 
indefinitely  until  required. 

3.  The  staining  of  the  sections.— In  order  to  prepare 
the  section  for  staining  and  to  stain  it  a  series  of  simple 
reagents  are  required.  These  may  be  poured  out  into  special 
staining  tanks  if  a  number  of  sections  are  being  treated  and 
the  sections  can  be  completely  immersed  in  the  tanks.  If  few 
sections  only  are  being  prepared  it  is  sufficient  to  pour  the 
reagents  over  each  slide,  but  care  must  be  taken  to  cover  the 
slide  completely  and  to  renew  the  reagent  if  there  is  danger 
of  evaporation. 

To  prepare  the  section  for  staining  proceed  as  follows : — 

Cover  with  xylol,  and  leave  till  the  paraffin  is  completely 
dissolved  and  the  section  is  perfectly  clear  and  transparent. 

Drain  off  the  xylol. 

Cover  with  absolute  alcohol  for  2  minutes.  (The  section 
becomes  opaque.) 

Cover  with  methylated  spirit  for  2  minutes. 

Wash  in  water  for  2  minutes  and  leave  in  water  until 
required  to  stain. 

Should  the  section  come  off  the  slide  at  any  period  of  the 
process,  it  can  be  transferred  with  a  section  lifter  through  the 
intermediate  reagents  to  water,  floated  in  a  beaker  of  water,  and 
picked  up  again  on  a  clean  slide.  The  water  on  the  slide  is 
drained  off  so  far  as  possible,  and  the  section  is  blotted  very 
firmly  on  the  slide  with  clean,  dry  filter  paper. 

The  preparation  of  the  various  staining  reagents  is  given  in 
Chapter  XIII. 


HISTOLOGICAL   METHODS.  431 

A.  To  stain  with  Hsemalum  and  Eosin. — This  is  the 
most  useful  routine,  stain  and  is  sufficient  for  all  ordinary 
purposes. 

Eemove  from  water  and  drain  off  excess  of  water. 

Filter  the  haemalum  on  to  the  section  and  control  the  depth 
of  stain  by  examining  the  section,  after  washing  it  in  tap  water, 
under  the  low  power  of  the  microscope.  The  period  of  staining 
differs  for  different  samples  of  haemalum  and  for  different 
tissues.  Three  minutes  is  the  time  for  the  majority  of  stains 
and  tissues.  Very  cellular  tissues,  such  as  a  lymph  gland, 
require  a  shorter  time  and  necrotic  tissues  a  longer  time.  If 
in  doubt  it  is  preferable  to  understain  the  section  and  then  to 
return  it  to  the  haemalum  for  another  period. 

Leave  in  tap  water  till  the  section  is  quite  blue. 

Cover  with  2  per  cent,  watery  eosin  for  20  seconds. 

Dip  in  tap  water. 

Drain  off  water  and  cover  with  methylated  spirit  for 
1  minute. 

Drain  off  methylated  spirit  and  cover  with  absolute  alcohol 
for  3  minutes. 

Drain  off  alcohol  and  cover  with  xylol  for  3  minutes. 

Wipe  off  the  xylol  from  the  slide  in  the  neighbourhood  of 
the  section,  but  do  not  allow  the  section  to  dry. 

Mount  in  Canada  balsam. 

The  cells  and  nuclei  are  stained  blue  and  the  connective 
tissue  pink. 

For  diagnostic  purposes  it  is  often  advantageous  to  stain  one 
section  with  haemalum  only.  A  single  stain  frequently  gives  a 
better  idea  of  the  nature  of  a  doubtful  tumour  than  a  brightly 
differentiated  one. 

B.  To  stain  with  Hsemalum  and  van  Gieson  :— 
After  staining  in  haemalum,  soak  in  water. 

Cover  with  van  Gieson' s  stain  for  20  seconds. 

Wash  very  rapidly  in  water. 

Wash  very  rapidly  in  methylated  spirit. 

Place  in  absolute  alcohol  for  3  minutes. 

Place  in  xylol.     Mount  in  Canada  balsam. 

Fibrous  tissue  is  stained  pink,  muscle  brown,  and  elastic 
tissue  yellow.  The  haemalum  staining  should  be  rather 
deep,  otherwise  the  picric  acid  will  stain  the  cell  protoplasm 
yellow. 


432  CLINICAL   PATHOLOGY. 

C.  To  stain  with  carbol-thionin  :— 

Cover  the  slide  with  carbol-thionin  for  5  minutes. 

Wash  rapidly  in  water. 

Wash  in  methylated  spirit  for  about  1  minute,  observing  the 
section  from  time  to  time  under  the  microscope,  and  stopping 
as  soon  as  any  pink  colour  appears. 

Place  in  absolute  alcohol  for  2  minutes. 

Place  in  xylol,  and  mount  in  Canada  balsam. 

Fibrous  tissue  is  stained  red  and  cells  purple.  Micro- 
organisms are  well  shown  and  stain  purple.  Fibrin  can  be 
stained  in  this  way.  The  method  is  not  recommended  for 
permanent  preparations,  since  the  stain  is  liable  to  gradually 
dissolve  out  into  the  mountant,  but  is  useful  as  a  rapid  and 
simple  stain  for  organisms  in  inflammatory  tissues. 

D.  To  stain  for  tubercle  bacilli  in  section  :— 
Take  the  slide  from  xylol  through  spirit  to  water. 
Filter  boiling  carbol-fuchsin  on  to  the  slide. 
Stain  for  7  minutes  with  3  changes  of  fuchsin. 
Dip  in  water. 

Wash  in  25  per  cent,  sulphuric  acid  till  decolorised. 

Wash  in  water. 

If  the  red  colour  comes  back  return  to  acid. 

Eepeat  until  the  tissue  after  3  minutes'  washing  in  fresh 
water  is  colourless,  or  at  the  most  of  a  very  pale  pink  colour. 

Stain  with  hsemalum.     Soak  in  water  till  blue. 

Take  back  through  alcohol  and  xylol,  and  mount  in  Canada 
balsam. 

The  tubercle  bacilli  are  stained  red  and  the  tissues  blue. 

Leprosy  bacilli  are  stained  in  the  same  manner,  but  12  per 
cent,  acid  is  used  to  decolorise  and  a  distinct  pink  is  left  in 
the  tissue. 

E.  To  stain  by  Gram's  method  in  section  :— 
Take  the  slide  from  xylol  through  spirit  to  water. 

Filter  freshly-prepared  aniline  gentian-violet  (see  page  150), 
on  to  the  slide  for  10  minutes. 

Dip  in  water. 

Cover  with  Gram's  iodine  for  1  minute. 

Wash  in  methylated  spirit  until  the  colour  ceases  to  come 
out  freely  and  only  a  faint  blue  is  left  in  the  section. 

Wash  in  water. 

Stain  in  J  per  cent,  safranin  (filtered)  for  20  seconds. 


HISTOLOGICAL  METHODS.  433 

Wash  in  water. 
Dip  in  methylated  spirit. 
Place  in  absolute  alcohol  for  2  minutes. 
Clear  in  xylol,  and  mount  in  Canada  balsam. 
Gram-positive    organisms    are    stained    blue    and    Gram- 
negative  red. 

F.  To  stain  with  Leishman's  stain  in  section  :— 
Clear  in  xylol. 

Place  in  absolute  alcohol  for  2  minutes. 

Wash  in  distilled  water. 

Cover  with  Leishman's  stain,  and  add  immediately  double 
the  volume  of  distilled  water.     Stain  for  7  minutes. 

Wash  in  distilled  water. 

Wash  in  1  in  1500  acetic  acid  in  distilled  water,  observing 
the  section  from  time  to  time  under  the  microscope  until 
the  connective  tissue  appears  pink. 

Wash  in  distilled  water. 

Stain  in  a  saturated  solution  of  eosin  in  absolute  alcohol  for 
20  seconds. 

Wash  in  absolute  alcohol. 

Clear  in  xylol,  and  mount  in  Canada  balsam. 

The  method  is  to  be  used  as  a  differential  stain  for  cells. 

G.  To  stain  for  free  iron  in  section : — 
Take  sections  through  to  water. 

Place  in  2  per  cent,  potassium  ferrocyanide  in  distilled 
water  for  1  hour. 

Place  in  1  per  cent,  acid  alcohol  for  30  minutes.  The  acid 
alcohol  has  the  composition — 

1  c.c.  strong  HC1. 
70   ,,  absolute  alcohol. 
29   „  distilled  water. 
Eeturn  through  methylated  spirit  to  xylol,  and  mount  in 
Canada  balsam. 

Free  iron  pigment  is  stained  a  greenish  blue. 
The   tissue  can   be   lightly   counterstained   with   eosin   or 
safranin  if  desired. 


P.  'AS 


INDEX. 


Abscess,  116,  165 

pulmonary,  367 
tropical,  blood  changes  in, 
19 
Acarus  scabiei,  352 
Aceto-acetic  acid,  244 
Acetone,  244 

Acholuric  family  jaundice,  28 
Achorion  Schonleinii,  146,  355 
Achylia  gastrica,  295 
Acid,  aceto-acetic,  244 

"  active  "  hydrochloric,  294 
ammonium  urate,  257 
B-hydroxybutyric,  244 
-fast  bacillus,  123 
free  hydrochloric,  293 
glycuronic,  247 
lactic,  297 
Acidosis,  228 
Acne  bacillus,  130 
Acne  vulgaris,  165,  359 
Acquired    syphilis.    Wassermann   re- 
action in,  68 
Actinomyces,  144 
Actinomycosis,  371,  384 
Acute  inflammation,  376 
blood  changes  in,  17 
Adenoma,  392 

papillary,  393 
fibro-,  393 
Mrogenes  capsulalus  bacillus,  132 
Agar-agar,  106,  182 
Agar-oleic  acid,  183 

plate  cultures,  106 
slope  cultures,  106 
stab  cultures,  106 
Agglutinins,  48 

as  a  test  for  organisms,  57 
as  evidence  of  infection,  57 
in  diagnosis,  49 
in  dysentery,  56 
in  Malta  fever,  56 
in  paratyphoid  infections,  55 
in  typhoid  fever,  49 
Ague,  86 

Albumin  in  faeces,  321 
tests  for,  215 
Albuminometer,  Esbach's,  238 
Albuminuria,  239 
Albus  staphylococcus,  ¥15'' 


Alkalinity  of  the  blood,  97 

Wright's  method,  97 
Alkaptonuria,  224 
Alveolar  sarcoma,  415 
Ammonia  nitrogen,  228 
Amceba  coli,  340 
Amoebic  dysentery,  340 
Amorphous  phosphates,  258 

urates,  256 
Amyloid  casts,  255 

disease,  220 
Anaemia,  aplastic,  11 

infantum  pseudoleukaemica, 
27 

pernicious,  7,  8,  99 

secondary,  24 

splenic,  22 

splenic  of  infants,  27 

Von  Jaksch's,  27 
Anaerobic  cultures,  162 
Angeioma,  397 
Anginosus  streptococcus,  119 
Angular  conjunctivitis,  133 
Animal  inoculation,  109 
parasites,  329 

blood  changes  with, 
19 
Ankylostoma  americanum,  336 

duodenale,  336 
Anophelinae,  86 
Anthrax  bacillus,  130 
Anti-colon  bacillus  serum,  176 
Anti-diphtheritic  serum,  175 
Antiformin,  156 
Antigen,  66 

syphilitic,  72 
Anti-meningococcus  serum,  176 
Anti-plague  serum,  178 
Anti-pneumococcus  serum,  176 
Anti-sera,  174 
Anti-tetantic  serum,  175 
Appendicitis,  213 

blood  changes  in,  18 
Arabinose,  181 
Arthritis,  117 

gonorrhceal,  164 
rheumatoid,  208 
Ascaridae,  338 
Ascaris  lumbricoides,  338 
Aspergillosis,  350 

28—2 


436 


INDEX. 


Aspergillus  niger,  146 
Asthma,  367 

blood  changes  in,  20 
Aureus  staphylococcus,  115 
Autoclave,  178 
Avian  tubercle  bacillus,  125 

Bacilltjbia,  282 
Bacillus,  acid-fast,  123 
acne,  120 

cerogenes  capsulatus,  131 
anthrax,  130 
avian  tubercle,  125 
Bordet-Gengou,  80 
bovine  tubercle,  124 
butter,  125 
coli  communis,  137 
comma,  140 
diphtheria,  127 
Ducrey's,  278 
dysentery,  137 
enteritidis  (Gaertner),  136 
enteritidis  sporogenes,  107 
fish  tubercle,  125 
Flexner's,  137 
Friedlander's  pneumo-,  370 
Gaertner's,  136 
glanders,  110 
Hofmann's,  128,  130 
influenza,  132,  198 
Kitasato,  133 
Koch-Weeks,  133,  348 
lepra,  126 

malignant  oedema,  140 
mallei,  133 

Moeller's  timothy-grass,  125 
Morax-Axenfeld,  133,  348 

Morgan's  No.  1,  343 

paratyphoid  A,  136 

paratyphoid  B,  136 

pestis,  134 

Pfeiffer's,  132 

plague,  372 

proteus,  139 

proteus  in  urine,  282 

pyocyaneus,  139 

Rabinowitch's  butter,  125 

saprophytic,  198 

Shiga,  137 

smegma,  125 

subtilis,  123 

suipestifer,  136 

tetanus,  130 

tubercle,  123 

typhosus,  133 

Vincent's  fusiform,  143 

whooping-cough,  131 

xerosis,  128,  347 
Bacteriology  of  the  bile,  305 

urine,  276 
Barber's  rash,  358 
Basophil,  coarsely  granular,  5 


Basophil,  finely  granular,  5 
Bed  bug,  354 
Bence-Jones  protein,  241 
B-hydroxybutyric  acid,  230,  344 
Bile,  304,  319 

bacteriology  of,  305 
Bilharzia  hsematobia,  255,  330 
Biuret  reaction,  325 
Blastomyces,  355 
Blepharoblast,  89 
Blood,  alkalinity  of,  97 

Wright's  method,  97 
Blood  changes  in — 

acute  inflammation,  17 
animal  parasites,  19 
appendicitis,  18 
cachexia,  26 
carcinoma,  19,  21 
cardiac  failure,  23 
chicken-pox,  20 
children,  26 

chronic  inflammation,  21 
congenital  morbus  cordis,  24 
congenital  syphilis,  29 
gonorrhoea,  21 
haemorrhage,  25 
infantile  scurvy,  30 
influenza,  22 
lead  poisoning,  26 
malaria,  22 
measles,  22 
metallic  poisons,  26 
pneumonia,  17 
purpura,  30 
rickets,  29 
scarlet  fever,  20 
skin  lesions,  20 
small-pox,  20 
spasmodic  asthma,  20 
spring  catarrh,  20 
syphilis,  21 
tropical  abscess,  19 
tuberculosis,  19 
typhoid  fever,  22 
Blood  cultures,  81 

in  rheumatic  fever,  81 
in  typhoid  fever,  83 
Blood  films,  to  make,  44 
fresh,  3 

in  test  meal,  299 
in  urine,  249 
normal,  3 

oxygen  content  of,  98 
parasitology  of,  81 
platelets,  3,  7 
primary  diseases  of,  7 
secondary  diseases  of,  17 
serum,  Loffler's,  185 
serum  medium,  184 
specific  gravity  of,  96 
spectroscopic  examination  of,  92 
to  obtg.jn,  33 


INDEX. 


437 


Blood,  unstained,  34 

viscosity  of,  47 
Boils,  165,  358 
Bordet-Gengou  reaction,  67 
Bothriocephalus  latus,  331 
Branchial  cyst,  419 
Brevis  streptococcus,  119 
Bronchial  fluke,  330 
Bronchiectasis,  165 
Broth,  105 

glucose,  181 

glycerine,  181 

litmus  carbohydrate,  107 

media,  180 

neutral  red,  108,  181 
Buchner's  tube,  162 

Cachexia,  blood  changes  in,  26 
Calcareous  degeneration,  389 
Calcium  carbonate,  259 

oxalate,  259 
Calculous  anuria,  220 
Callosity,  391 
Calmette's  reaction,  173 
Cambridge  microtome,  429 
Cammidge's  method     of     estimating 
fats,  321 
pancreatic  reaction,  273 
Capsules,  103 

to  stain,  162 
Carbol  fuchsin,  187 

-thionin  tissue  staining,  432 
-thionin,  to  stain  with,  149 
Carboluria,  225 
Carbonic  oxide  poisoning,  92 
Carboxyhaemoglobin,  92 
Carbuncles,  358 
Carcinoma,  403 

blood  changes  in,  19 
columnar-celled,  410 
encephaloid,  404 
of  stomach,  295 
scirrhous,  404 
spheroidal-celled,  408 
squamous-celled,  404 
Cardiac  failure,  blood  changes  in,  23 
Casts,  253 

amyloid,  255 
cellular,  254 
granular,  254 
hyaline,  255 
prostatic,  250 
uratic,  253 
Cell,  endothelial,  192 
endothehoid;  377 
epithelioid,  378 
giant,  380 

giant,  of  Virchow,  386 
lymphoid,  191,  377 
malignant,  193 
plasma,  378 
Cell-nest,  405 


Cellular  casts,  253 
CeUulitis,  118 
Centrifugal  machine,  63 
Cerebro-spinal  fluid,  203 

Wassermann  reaction  in,  69,  77 
Cestoda,  329,  331 
Chancre,  142 

soft,  278 
Charcot-Leyden  crystals,  366 
Chicken-pox,  blood  changes  in,  20 
Children,  blood  changes  in,  26 
Chlorides,  estimation  of,  272 
Chloroma,  15 
Chlorosis,  8,  98 
Cholsemia,  95 

congenital  family,  28 
Cholelithiasis,  296 
Cholera,  141 

red  reaction,  141 
vibrio,  105,  140,  343 
Cholesterin  crystals,  307 
Chondroma,  396 
Chorion  epithelioma,  417 
Chronic  gastritis,  296 

inflammation,  blood  changes 
in,  21 
Chylous  fluid,  203 
Gitreus  staphylococcus,  115 
Clonorchis  sinensis,  329 
Cloudy  swelling,  387 
Coagulation  time,  46 
Coffin-lid  crystal,  258 
Coli  communis  bacillus,  137 
Colitis,  ulcerative,  137 
Colloid  degeneration,  388 
Colon  bacillus  in  urine,  137,  279 
Colorimeter,  Duboscq,  270 
Colour  index,  4 

Columnar-celled  carcinoma,  410 
Comma  bacillus,  140 
Complement,  66 

fixation  test  in — ■ 
gonorrhoea,  65 
hydatid  disease,  65 
tuberculosis,  65 
Condenser,  paraboloid,  161 
Congenital  family  cholsemia,  28 

syphilis,    Wassermann   re- 
action in,  69 
Coniferin,  181 
Conjunctival  sac,  347 
Conjunctivitis,  angular,  133 

diplo  -  bacillary,    133, 

348 
membranous,  349 
Cornea,  350 
Corneal  ulcer,  350 
Cornet's  forceps,  147 
Corpuscles,  red,  3 

white,  4 
Cover-glasses,  to  clean,  42 
Crab  louse,  353 


438 


INDEX. 


Creatine,  269 

Creatinine,  269 

Gulex  fatigans,  91 

Cultural  characters,  104 

Culture,  blood,  81 
plate,  106 
slope,  106 
stab,  106 

Cultures,  anaerobic,  162 

Curschmann's  spirals,  365 

Cylindroma,  401 

Cysticerus  bovis,  333 

cellulosce,  333 

Cystine,  259 

Cystoma,  419 

Cysts,  209,  418 

branchial,  419 
degeneration,  420 
dermoid,  211,  410 
hydatid,  209,  193 
mesenteric,  211 
other  abdominal,  211 
ovarian,  211 
pancreatic,  210 
ranula,  419 
renal,  211 
retention,  419 
retroperitoneal,  211 
sebaceous,  419 

Cyto-diagnosis,  191 


Decalcification  of  tissue,  428 
Decalcifying  fluid,  428 
Deciduoma  malignum,  417 
Degeneration,  387 

calcareous,  389 

colloid,  388 

fatty,  389 

granular,  10 

hyaline,  388 

lardaceous,  388 

polychromatophilic,  4 
Dental  osteoma,  397 
Dentium,  spirochceta,  143 
Dermoid  cyst,  211,  419 
Desmoid  test,  299 
Dextrose,  181 
Diabetes,  220 

insipidus,  220 
Diacetic  acid,  230 
Diagnosis,  agglutinins  in,  49 

Wassermann    reaction    in, 
68 
Diastase,  210 
Dibothriocephaloidea,  331 
Differential  count,  45 
Diphtheria,  129,  288 

bacillus,  109,  127 
nasal,  363 
Diphtheroid  bacillus,  109,  128 
Diplo-bacillary  conjunctivitis,  348 


Diplococcus    intracellularis    meningi- 
tidis, 121 
Frankel's,  116 
lanceolatus,  116 
rheumaticus,  119 
Diplogonoporus  grandis,  332 
Dorset's  egg  medium,  157,  184 
Dracunculus,  354 
Duboscq  colorimeter,  270 
Ducrey's  bacillus,  278 
Duodenal  ulcer,  31,  296 
Dysentery,  137 

amoebic,  340 
bacillus,  137 
Widal  reaction  in,  49 
Dyspepsia,  289 

ECCHONDROMA,  396 

Edestin  method,  297 

Emphysematous  gangrene,  131 

Empyema,  117,  196 

Encephaloid  carcinoma,  404 

Enchondroma,  396 

Endemic  haemoptysis,  368 

Endocarditis,  infective,  81,  119,  164 

Endothelial  cell,  192 

Endothelioid  cell,  377 

Endothelioma,  401 

Endotoxins,  167 

Entamoeba  coli,  340 

histolytica,  340 

Enteritidis  bacillus,  136 

sporogenes  bacillus,  107 

Enteritis,  infantile,  137 

Envelope  crystals,  259 

Eosin,  188 

Eosinophil  cell,  5,  193 

Eosinophilia,  19 

Epithelial  cells  in  urine,  251 

Epithelioid  cell,  378 

Epithelioma,  404 

chorion,  417 

Epulis,  394 

Erysipelas,  118,  357 

Erythremia,  23,  24 

Erythrasma,  355 

Esbach's  albuminometer,  238 
reagent,  238 

Estimation  of  chlorides,  272 

phosphates,  271 
purines,  268 
sulphates,  272 
uric  acid,  266 

Ewald's  test  meal,  290 

Examination  of  throat,  129 

Exudates,  199 

tuberculous,  198 

Eyre's  scale,  181 

Fcecalis,  streptococcus,  118,  119 
Faeces,  abnormal  ingredients,  317 
albumin  in,  321 


INDEX. 


439 


Faeces,  amount  of,  316 

bacteriology  of,  341 

banana  fibres  in,  317 

bile  in,  318 

chemical  examination  of,  318 

colour  of,  316 

consistency  of,  317 

crystals  in,  327 

elastic  fibres  in,  326 

epithelium  in,  327 

fat  in,  321 

gall-stones  in,  318 

intestinal  sand  in,  318 

microscopical  investigation  of, 
325 

mucin  in,  324 

mucous  casts  in,  317 

muscle  fibres  in,  326 

naked- eye  examination  of,  316 

occult  blood  in,  318 

odour  of,  317 

peptone  in,  324 

tubercle  bacilli  in,  157 
Fasciolopsis  buski,  329 
Eat,      estimation      of,      Cammidge's 
method,  321 

in  fseces,  321 
Fatty  degeneration,  389 
Favus,  146 

Fehling's  solution,  217 
Fever,  relapsing,  90 
Fibrin,  3,  265 
Fibro-adenoma,  393 
Fibroblast,  378 
"  Fibroids."  394 
Fibroma,  394 
Fibro-myoma,  394 
Fibro-neuroma,  394 
Filaria  diurna,  91 
noclurna,  90 
perstans,  91 
Filarise,  255 
Filariasis,  90 
FilariidEe,  335 

Finkler-Prior  vibrio,  141,  344 
Fish  tuberculosis,  125 
Fixation  of  tissues,  426 
Flagella,  to  stain,  163 
Flea,  354 
Flexner's  bacillus,  137 

serum,  176 
Fluid,  cerebro-spinal,  203 

chylous,  203 

decalcifying,  428 

opalescent,  202 

pericardial,  201 

pleural,  196 

pseudochylous,  203 

spermatocele,  208 

synovial,  207 

Zenker's,  428 
Follicular  impetigo,  357 


Follicular  tonsilitis,  288 
Forceps,  147 

Cornet's,  147 
Frankel's  diplococcus,  116 
Free  hydrochloric  acid,  293 

iron  in  section,  433 
Friedlander's  pneumo-bacillus,  370 
Fungus,  ray,  144 
Fusiform  bacillus  of  Vincent,  143 


Gaertner's  bacillus,  136 

Gall-stones,  306 

Gangrene,  emphysematous,  131 

pulmonary,  367 
Gastric  juice,  289 

ulcer,  296,  311 
Gastritis,  chronic,  296 
Gelatin,  106,  183 
Gentian  violet,  187 
Gerhardt's  reaction,  244 
Gerrard's  method,  242 
solution,  243 
ureometer,  232 
Giant  cell,  380 

of  Virchow,  386 
Giemsa's  stain,  160,  187 
Glanders,  133,  384 

bacillus,  110 
Glioma,  399 
Globulin,  206  _ 
Glossina  morsitans,  88 

palpalis,  88 
Glucose  broth,  181 

in  urine,  241 
tests  for,  216 
Glycerine  broth,  183 
Glycogensemia,  94 
Glycosuria,  243 
Glvcuronic  acid,  247 
Gmelin's  test,  96,  223 
Goat's  milk,  120 
Gonococcus,  82,  121,  276 
Gonorrhoea,  122,  278 

blood  changes  in,  21 
complement  fixation  test, 
65 
Gonorrhoeal  arthritis,  164 
Gracilis  spirochceta,  143 
Gram's  iodine,  187 

method,  103,  150 

in  section,  432 
Granular  bodies,  141 
casts,  254 
degeneration,  10 
vaginitis,  114 
Granuloma,  375 
Gravel,  257 

Griinbaum-Widal  reaction,  49 
Guiac  test,  222 
Guinea-worm,  354 
Gum  sections,  422 


440 


INDEX. 


Gum  solution,  422 
Giinzburg's  test,  291 

Hemagglutinins,  30,  48,  58 

Hsemalum,  188 

and  eosin  stain,  431 

Hsemangeioma,  397 

Hsematoporphyrinuria,  224 

Hsemochromatosis,  352 

Hsemocytometer,  39 

Haemoglobin,  4,  34 

reduced,  92 

Haemoglobinometer,  34 

Haldane's,  34 
Oliver's,  36 
Tallqvist's,  34 

Hemoglobinuria,  222 

Haemolysis,  65 

Haemophilia,  28 

Haemoptysis,  endemic,  368 

Haemorenal  index,  237 

Haemorrhage,  blood  changes  in,  25 

Hanging  drops,  5 

Harvest  bug,  354 

Hay  fever,  363 

Hay's  test,  223 

Hecht-Fleming  modification,  75 

Heller's  test,  216 

Histological  methods,  421 

Hodgkin's  disease,  22,  384 

Hofmann's  bacillus,  128,  130 

Hot-air  steriliser,  177 

Hyaline  casts,  255 
cell,  5 
degeneration,  388 

Hydatid  cyst,  193,  209 

disease,  complement  fixation 
test,  65 

Hydrocele,  208 

Hydrochloric  acid,  "  active,"  294 
free,  294 

Hyperchlorbydria,  296 

Hypernephroma,  417 

Hyphomycetes,  145 

Idiopathic  peritonitis,  314 
Immunity  unit,  174 
Impetigo  contagiosa,  357 

follicular,  357 
Incubator,  148 
Index,  opsonic,  165 
Indian-ink  method,  160 
Indole  reaction,  105 
Infantile  enteritis,  137 

scurvy,  blood  changes  in,  11 
Infection,  agglutinins  as  evidence  of,  57 
Infective  endocarditis,  81,  119,  164 

vaccine  treatment  in,  83 
Inflammation,  375 

acute,  376 

blood      changes 
in,  17 


Inflammation,  chronic,  blood  changes 

in,  21 
Influenza  bacillus,  132,  198 

blood  changes  in,  22 
Inoculation,  prophylactic,  136 
Inspissator,  178 
Intestinal  obstruction,  314 

toxaemia,  286 
Intracellular  toxins,  167 
Inulin,  181 
Involution  forms,  127 
Iodine  solution,  84 

test,  223 
Iodophilia,  94 
Iodopin  test,  300 
Itch,  352 
Ivory  osteoma,  397 

Jat/ndice,  acholuric  family,  28 
Jenner's  stain,  43,  45 

Kala-azae,  89 
Keratoma,  391 
Kitasato  bacillus,  133 
Kjeldahl's  method,  230 
Klebs-Loffler  baciUus,  127 
Koch's  old  tuberculin,  173 

vibrio,  344 
Koch-Week's  bacillus,  133 

Lachrymal  sac,  350 
Lactic  acid,  297 
Lactose,  181,  246 
Lardaceous  degeneration,  288 
Latent  syphilis,  Wassermann  reaction 

in,  69 
Lead  poisoning,  blood  changes  in,  26 
Leiomyoma,  394,  395 
Leishman's  stain,  43,  45 

in  section,  433 
Leishman- Donovan  body,  89 
Leishmania,  89 
Lepra  bacillus,  126 
Leprosy,  126,  363,  383 
Leptus  autumnalis,  353 
Leucine,  260 
Leucocytes,  5 

enumeration  of,  40 

origin  of,  6 

Strong's  method  for,  40 

Thoma-Zeiss  method  for, 
41 
Leucocytosis,  4,  .17 
Leucopenia,  21 
Leukaemia,  lymphoid,  13,  15 

myeloid,  7,  13 
Lipaemia,  93 
Lipase,  210 
Lipoids,  67 
Lipoma,  396 
Lipuria,  227 


INDEX. 


441 


Litmus  carbohydrate  broth,  107,  181 
milk,  107,  183 
solution,  181 
Liver  abscess,  269,  304 

flukes,  329 
Lobar  pneumonia,  117 
Loffler's  blood  serum,  185 

methylene  blue,  to  stain,  186 
Lumbar  puncture,  203 
Lung  puncture,  201,  372 
Lymphangeioma,  397 
Lymphangitis,  116,  358 
Lymphocytes,  large,  5 

small,  5,  191 
Lymphoid  cell,  191,  377 
leukaemia,  13 

chronic,  15 
Lymphoma,  394 

MacConkey's  medium,  108,  182 
Madura  foot,  145,  355 
Malaria,  86 

blood  changes  in,  22 
Malarial  parasites,  86 
Malignant  cells,  193 

endocarditis,  81 
oedema  bacillus,  149 
pustule,  130 
Mallei  bacillus,  133 
Mallein,  173 
Malta  fever,  120 

agglutinins  in,  56 
Maltose  agar  medium,  145 
Mannite,  181 
Mast  cell,  5 

Mayhew's  ureometer,  234 
"  Measled  "  pork,  332 
Measles,  blood  changes  in,  22 
Meckel's  cartilage,  396 
Media,  standardisation  of,  181 

sterilisation  of,  176 
Medium,  blood  serum,  184 

broth,  180 

Dorset's  egg,  157,  184 

MacConkey's,  182 

maltose  agar,  145 

ox  bile,  185 

potato,  184 
Megaloblasts,  4,  10 
Melaninuria,  225 
Melanotic  sarcoma,  416 
Meningitis,  117,  206 
Meningococcus,  121,  205 
Metallic  poisons,  blood  changes  in,  26 
Metchnikoff's  vibrio,  141 
MethsemoglobinEemia,  92 
Methylene  blue,  186 

Loffler's,  187 
Micrococcus  catarrhalis,  120 
melitensis,  120 
Microscope,  32 

ultra,  161 


Microsporon  Audouini,  145,  354 
furfur,  146,  355 
minutissimum,  355 
Microtome,  Cambridge,  429 
Williams's,  422 
Miliary  tubercle,  379 
Milk,  goat's,  183 
litmus,  183 
Millon's  reagent,  241 
Minimum  lethal  dose,  174 
Moeller's  timothy-grass  bacillus,  125 
Mole,  398,  416 
Molluscum  contagiosum,  387 

fibrosum,  386,  394 
Morax-Axenfeld  bacillus,  133 
Morbus     cordis,     congenital,      blood 

changes  in,  24 
Morgan's  No.  1  bacillus,  343 
Mucin,  241 

Mucoid  degeneration,  387 
Mucous  polyp,  394 
Mulberry  calculi,  262 
Murexide  test,  264 
Myeloblast,  6,  13 
Myelocytes,  6 
Myeloid  leuksemia,  4,  7 

acute,  13 
sarcoma,  414 
Myoma,  395 

fibro-,  394 
Myxoma,  396 
Myxo-sarcoma,  396 

NiEVi,  397 
Nagana,  88 
Nasal  catarrh,  362 

diphtheria,  363 
Nasgar,  109,  183 
Neisser's  method  of  staining,  159 
Nematodes,  334 
Nephritis,  acute,  231 

chronic  interstitial,  231 
Neuro-fibroma,  394 
Neuroma,  400 

plexiform,  400 
Neutral  red  broth,  108,  181 
Neutrophils,  polynuclear,  192 
Nits,  353 

Nonne  and  Apelt's  method,  206 
Normoblasts,  4 
Nylander's  reagent,  217 
test,  217 

Oat- celled  sarcoma,  413 
(Edema,  malignant,  bacillus  of,  140 
Oidium  albicans,  146 
Oleic  acid  agar,  183 
Oligocythsemia,  24 
Oncosphere,  331 
Opalescent  fluids,  203 
Ophthalmia  neonatorum,  123 
Oppler-Boas  bacillus,  302 


442 


INDEX. 


Opsonic  index,  59,  167 

modification  of,  63 

value  of,  62 
Opsonins,  nature  of,  58 
Oral  sepsis,  286 
Orcinol  reaction,  247 
Organisms,  agglutinins  as  a  test  for, 

57 
Oriental  sore,  90 
Osazone  reaction,  246 
Osier's  disease,  24 
Osteo-arthritis,  286 
Osteoma,  397 

dental,  397 
ivory,  397 
Osteomyelitis,  116 
Osteosarcoma,  412 
Ovarian  cysts,  211 
Ox  bile  medium,  185 
Oxygen  content  of  blood,  98 
Oxyhaemoglobin,  92 
Oxyphil,  coarsely  granular,  5 

finely  granular,  5 
Oxyuris  vermicularis,  339 


Pallida,  spirochoeta,  141 

Pallidum,  treponema,  141 

Pancreatic  efficiency,  303 
reaction,  273 

Papillary  adenoma,  393 

Papilloma,  391 

Paraboloid  condenser,  161 

Paraffin  sections,  426 

Paragonimus  westermani,  330,  368 

Parasitology  of  the  blood,  81 

Parasyphilis,  Wassermann  reaction  in, 
69 

Paratyphoid  bacillus,  136 

infections,  agglutinins  in, 
55 

Parotid  tumour,  401 

Pediculi,  353 

Pemphigus  neonatorum,  357 

Pentose,  246 

Pepsin,  297 

Pericardial  fluid,  201 

Peritoneal  fluid,  202,  311 

Peritonitis,  117 

idiopathic,  314 
post-operative,  314 
tuberculous,  315 

Pernicious  anaemia,  7,  8,  99 

Pertenuis,  spirochceta,  143 

Pertussis,  132 

Pestis,  bacillus,  134 

Petri  dish,  154,  185 

Pettenkofer's  test,  224,  319 

Pfeiffer's  bacillus,  132 
reaction,  110 

Phloroglucinol  reaction,  246 

Phosphates,  amorphous,  258 


Phosphates,  estimation  of,  271 
stellar,  258 
triple,  258 
Pigmentation,  389 
Pipettes,  Strong's,  38 
to  clean,  42 
Pityriasis  versicolor,  146 
Plague,  134 

bacillus,  372 
Plasma  cell,  378 
Plate  cultures,  106 
Platinum  wire,  147 
Pleural  fluid,  196 
Plexif orm  neuroma,  400 
Pneumococcus,  82,  116,  196,  370 
Pneumoconiosis,  368 
Pneumonia,  blood  changes  in,  17 

lobar,  117 
Poikylocytosis,  9 
Poisoning,  carbonic  oxide,  92 
Poisons,  lead,  blood  changes  in,  26 

metallic,  blood  changes  in,  26 
Polar  staining,  134 
Polychromatophilic  degeneration,  4 

Polycythemia,  4,  23 

splenic,  11,  24,  99 

Polvnuclear  neutrophils,  192 

Polypi,  393 

Post-operative  peritonitis,  314 

Potato  medium,  184 

Prior  vibrio,  141 

Proglottis,  331 

Prophylactic  inoculation,  136 

use  of  vaccines,  172 

Prostatic  casts,  250 

Prostatitis,  278 

Proteids  in  urine,  237 

Protein,  Bence-Jones,  241 

Proteoses,  240 

Proteus,  bacillus,  139 

Psammoma,  401 

Pseudochylous  fluid,  203 

Pseudo-glioma,  350 

Puerperal  septicaemia,  119,  164 

Pulmonary  abscess,  367 
gangrene,  367 

Puncture  lumbar,  203 
lung,  201 

Purines,  estimation  of,  268 

Purinometer,  Walker-Hall,  269 

Purpura,  blood  changes  in,  30 

Pus  in  urine,  250 

tubercle  bacilli  in,  157 

Pyaemia,  116 

Pyocyaneus,  bacillus,  139 

Pyogenes,  streptococcus,  118 

Pyorrhoea  alveolaris,  119,  165,  287 

Pyosalpinx,  123,  313 

Rabin owitch's  butter  bacillus,  125 
Raffinose,  181 
Ranula,  419 


INDEX. 


443 


Ray  fungus,  144 
Reaction,  biuret,  325 

Calmette,  173 

cholera  red,  105,  141 

Gerhardt's,  244 

indole,  105 

orcinol,  247 

osazone,  246 

pancreatic,  Cammidge's,  275 

Pfeiffer's,  110 

phloroglucinol,  246 

Salkowski's,  307 

Von  Pirquet,  173 
Reagent,  Millon's,  241 

Nylander's,  217 
Reagents,  staining,  to  prepare,  186 
Recurrentis,  spirochceta,  144 
Red  cells,  3 

enumeration  of,  37 

fragility  of,  46 

sensitised,  66 

Strong's  method,  37 

Thoma-Zeiss  method,  38 
Reduced  haemoglobin,  92 
Eefringens,  spirochwta,  143 
Relapsing  fever,  90 
Renal  cysts,  210 

tumours,  417 

hypernephroma,  417 
von  Grawitz,  417 
Rennin,  298 
Retention  cyst,  419 
Retinal  glioma,  400 
Rhabdomyoma,  395 
Rheumatic  fever,  208 

blood  cultures  in,  81 
Rheumatoid  arthritis,  208 
Rickets,  blood  changes  in,  29 
Ringworm,  145,  354 
Rodent  ulcer,  406 
Rothera's  test,  245 
Round-celled  sarcoma,  412 

SAP.OTTitA'rJD's  maltose  agar  medium, 

145 
Saccharose,  181 
Safranin,  187 
Sahli's  desmoid  test,  299 
Salicin,  181 

Salivarius,  streptococcus,  118,  119 
Salkowski's  reaction,  307 
Salpingitis,  117 
Salt  formalin  solution,  425 
Saprophytes,  111 
Saprophytic  bacillus,  198 
SarcinEe,  123 
Sarcoma,  411 

alveolar,  415 

melanotic,  416 

myeloid,  414 

oat-celled,  413 

osteo-,  412 


Sarcoma,  round-celled,  412 

spindle-celled,  413 
Scale,  Eyre's,  181 
Scarlet  fever,  blood  changes  in.  20 
Scharlach  R.,  188 
Schistosomum  japonicmn,  331 
Schmidt-Werner  tube,  322 
Scirrhous  carcinoma,  404 
Sclerosis,  posterior  lateral,  11 
Scolex,  331 

Scurvy,  infantile,  blood  changes  in,  29 
Sebaceous  cyst,  419 
Sections,  examination  of,  374 
free  iron  in,  433 
Gram's  method,  432 
gum,  422 

Leishman's  stain  in,  433 
paraffin,  426 
staining  of,  430 
tubercle  bacilli  in,  432 
Sensitised  red  cells,  66 

vaccine  of  Besredka,  167 
Septicaemia,  82 

puerperal,  119,  164 
Serum,  albumin,  238 

anti-colon  bacillus,  175 
anti-diphtheritic,  175 
anti-meningococcus,  176 
anti -plague,  176 
anti-pneumococcus,  176 
anti-streptococcal,  175 
anti-tetanic,  175 
Flexner's,  51 
globulin,  238 
to  obtain,  51 
tube,  96 
uric  acid  in,  96 
Shiga  bacillus,  137 
Skin,  350 

bacteriology  of,  356 
lesions,  blood  changes  in,  20 
Slope  cultures,  106 
Small  lymphocytes,  191 
Small-pox,  blood  changes  in,  20 
Smegma  bacillus,  125 
Soft  chancre,  278 
Solution,  gum,  422 

salt  formalin,  425 
Sorbit,  181 
Spasmodic  asthma,  blood  changes  in, 

20 
Specific  gravity  of  blood,  96 
Spectroscope  test,  223 
Spectroscopic  examination  of  blood, 

92 
Spermatocele  fluid,  208,  209 
Spermatozoa,  209,  255 
Spheroidal-celled  carcinoma,  408 
Spindle-celled  sarcoma,  413 
Spirilla,  140 

Spirillum  obermeieri,  90 
of  Vincent,  143 


444 


INDEX. 


Spirochceta  dentium,  143 
gracilis,  143 
obermeieri,  144 
pallida,  141,  278 
pertenuis,  143 
recurrentis,  90,  144 
ref ring  ens,  143 
Spirochetosis,  90 
Spleen,  308 

puncture,  89 
Splenic  anaemia,  11 

of  infants,  27 
polycythsemia,  11,  24 
Spores,  103 

to  stain,  162, 163 
Sporotrichia,  355 

Spring  catarrh,  blood  changes  in,  20 
Sputum,  363 

bacteriology  of,  368 
elastic  fibres  in,  364 
fibrinous  casts  in,  366 
tubercle  bacilli  in,  155,  369 
Squamous-celled  carcinoma,  404 
Stab  cultures,  106 
Stain,  Giemsa's,  160,  187 

hsemalum  and  eosin,  431 
Jenner's,  43,  45 
Leishman's,  43,  45 
Van  Gieson's,  188,  431 
Staining  capsules,  162 

fat  in  tissues,  425 

flagella,  163 

polar,  134 

reagents,  to  prepare,  186 

sections,  430 

spores,  162 

with  carbol-thionin,  149 

with  Gram's  method;  150 

with     Loffler's     methylene 

blue,  159 
with  Neisser's  method,  159 
Standardisation  of  media,  181 
Staphylococci,  115 
Staphylococcus  albus,  115 
aureus,  115 
citreus,  115 
Steam  steriliser,  178 
Stellar  phosphates,  258 
Stercobilin,  316,  319 
Sterilisation  of  media,  177 
Steriliser,  hot-air,  177 
steam,  178 
Stock  vaccines,  165 
Stomach,  carcinoma  of,  295 
Streptococci,  118 

anginosus,  119 
brevis,  119 
fcecalis,  118,  119 
pyogenes,  82,  118 
salivarius,  118,  119 
Streptococcus,  118 
Strong's  method  for  red  cells,  37 


Strong's  pipettes,  38 

Strongylidse,  336 

Strongyloides  intestinalis,  334 

Sub-cultures,  to  make,  153 

Subtilis,  bacillus,  123 

Suipestifer,  bacillus,  136 

Sulph-hsemoglobinsemia,  93 

Sulphates,  estimation  of,  272 

Surra,  88 

Sycosis,  358 

Synovial  fluid,  207 

Syphilis,  142,  382 

blood  changes  in,  21 
congenital,  blood  changes  in, 
29 

Syphilitic  antigen,  72 

Syringo-myelia,  399 

Taenia  echinococcus,  333 
saginata,  333 
solium,  332 
Tseniidse,  331 
Teratoma,  418 
Test,  albumin,  215 

complement  fixation,  in  gonor- 
rhoea, hydatid  disease,  tuber- 
culosis, 65 
desmoid,  299 

Sahli's,  299 
Gmelin's,  96,  223 
guiac,  222 
Hay's,  223 
iodine,  223 
iodopin,  300 
murexide,  264 
Nvlander's,  217 
Pettenkofer's,  224,  319 
Rothera's,  245 
Sahli's  desmoid,  299 
Uffelmann's,  297 
Test  meal,  289 

blood  in,  299 
Ewald's,  290 
Tetanus  bacillus,  110,  130 

toxin,  130 
Thermos  flask,  148 
Thoma-Zeiss  method  for  red  cells,  38 
Thread  worms,  255 
Throat,  examination  of,  129 
Thrush,  146,  287 
Tissues,  decalcification  of,  428 
fixation  of,  426 
staining  fat  in,  425 
Toison's  fluid,  40 
Tonsilitis,  288 
Topfer's  solution,  291 
Total  acidity,  293,  294 
Toxins,  intracellular,  167 
Trachoma,  349 
Transudates,  199 
Trematoda,  329 
Treponema  pallidum,  141 


INDEX. 


445 


Trichineha  spiralis,  336 
Trichocephalus  dispar,  335 
Trichophyton  megalosporon  ectothrix, 

355 
endothrix, 
145,  354 
Trichotrachelidae,  335 
Triple  phosphates,  258 
Tropical  abscess,  blood  changes  in,  19 
Trypanosoma,  brucei,  88 
evansi,  88 
gambiense,  88 
lewisi,  88 
Trypsin,  210 
Tube,  Buchner's,  162 

Wright's,  51 
Tubercle  bacillus,  123 

in  fseces,  157 
in  pus,  157 
in  section,  432 
in  sputum,  155 
in  urine,  157,  279 
Tuberculides,  360 
Tuberculin,  167 

old,  Koch's,  173 
Tuberculosis,  blood  changes  in,  19,  21 
complement  fixation  test, 

65 
histology  of,  379 
Tuberculous  exudates,  198 

peritonitis,  315 
Tumours,  malignant — 
Alveolar  sarcoma,  415 
Carcinoma,  401 
Chorion  epithelioma,  417 
Columnar-celled  carcinoma,  410 
Deciduoma  malignum,  417 
Encephaloid  carcinoma,  404 
Endothelioma,  401 
Epithelioma,  404 
Hypernephroma,  417 
Melanotic  sarcoma,  416 
Myeloid  sarcoma,  414 
Oat-celled  sarcoma,  413 
Osteo-sarcoma,  412 
Rodent  ulcer,  406 
Round-celled  sarcoma,  412 
Sarcoma,  411 
Scirrhous  carcinoma,  404 
Spheroidal-celled  carcinoma,  408 
Spindle-celled  sarcoma,  413 
Squamous-celled  carcinoma,  404 
Teratoma,  418 
von  Grawitz  tumour,  417 
Tumours,  simple — 
Adenoma,  392 
Angeioma,  397 
Branchial  cyst,  419 
Callosity,  391 
Chondroma,  396 
Ecchondroma,  396 
Enchondroma,  396 


Tumours,  simple — 

Epulis,  394 

Fibro-adenoma,  393,  394 

Fibro-myoma,  394 

Fibro-neuroma,  394 

"  Fibroid,"  394 

Fibroma,  394 

Glioma,  399 

Hsemangeioma,  397 

Ivory  osteoma,  397 

Keratoma,  391 

Leiomyoma,  394,  395 

Lipoma,  396 

Lymphangeioma,  397 

Lymphoma,  394 

MoUuscum  fibrosum,  394 

Mole,  398 

Mucous  polyp,  394 

Myoma,  395 

Myxoma,  396 

Myxo-sarcoma,  396 

Nsevus,  397 

Neuro -fibroma,  394 

Neuroma,  399 

Osteoma,  397 

Papilloma,  391 

Parotid  tumour,  401 

Plexiform  neuroma,  399 

Polypus,  393 

Retinal  ghoma,  399 

Rhabdomyoma,  395 

Wart,  391 
Typhoid  bacillus  in  urine,  282 
carriers,  135,  306 
fever,  135,  161,  172 
agglutinins  in,  49 
blood  changes  in,  22 
blood  cultures  in,  83 
ulcer,  312 
Typhosus,  bacillus,  135 
Tyrosine,  260 

Ubtelmann's  test,  297 
Ulcer,  duodenal,  296,  312 
gastric,  296,  311 
typhoid,  312 
Ulcerative  endocarditis,  81 
Ultra-microscope,  161 
Uncinaria,  336 
Urates,  acid  ammonium,  257 

amorphous,  256 
Uratic  casts,  253 
Urea,  231 

Ureometer,  Gerrard's,  232 
Mayhew's,  234 
Ureteric  catheterisation,  213 
Urethritis,  122 
Uric  acid,  257 

estimation  of,  266 
in  serum,  96 
Urinary  calculi,  262 
casts,  253 


446 


INDEX. 


Urinary  deposits,  248 

Urine,  albumin  in,  215 
amount  of,  220 
bacillus  proteus  in,  282 
bacteriology  of,  276 
bile  in,  223 
blood  in,  221,  249 
casts  in,  253 
colon  bacillus  in,  279 
colour  of,  221 
deposits  in,  218 
eosin  in,  226 
epithelial  cells  in,  251 
glucose  in,  216,  241 
measurement  of,  214 
methylene  blue  in,  226 
naked-eye  appearance  of,  214 
phosphates  in,  226 
proteids  in,  237 
pus  in,  250 
reaction  of,  214 
routine  examination  of,  214 
specific  gravity  of,  214 
tests  for  albumin,  215 
tubercle  bacilli  in,  157,  279 
typhoid  bacilli  in,  282 
urates  in,  226 

Urinometer,  214 

Urobilin,  224 

Urostealith,  265 

Vaccines,  164 

diagnostic  use  of,  172 

method  of  preparation,  168 

of  Besredka,  167 

prophylactic  use  of,  172 

sensitised,  167 

stock,  165 

treatment  in  infective  en- 
docarditis, 83 
Vaginitis,  123 

granular,  114 
Van  Gieson's  stain,  188,  431 


Vaquez's  disease,  24 
Vibrio,  cholera,  140,  343 

Finkler-Prior,  141,  344 

Koch's,  344 

Metchnikoff,  141 
Vincent's  angina,  144,  288 

fusiform  bacillus,  143 
spirillum,  144 
Viscera,  bacterial  investigation  of,  161 
Volhard  process,  273 
Vomit,  301 

Von  Grawitz  tumour,  417 
Von  Jaksch's  anaemia,  27 
Von  Pirquet's  reaction,  173 


Walker-Hall  purinometer,  269 

Wart,  391 

Wassermann's  reaction,  65 

in  acquired  syphilis,  68 

in  cerebro-spinal  fluid,  69,  77 

in  congenital  syphilis,  69 

in  diagnosis,  68 

in  latent  syphilis,  69 

in  parasyphilis,  69 

in  response  to  treatment,  70 

technique  of,  71 
Whetstone  crystal,  257 
Whip-worm,  335 
Whitlow,  358 
White  corpuscles,  4 
Whooping-cough  bacillus,  132 
Widal's  reaction,  49 
Williams's  microtome,  422 
Wool-sorters'  disease,  131 

Xanthine,  265 
Xerosis  bacillus,  347 

Yaws,  360 

Zenker's  fluid,  428 
Ziehl-Neelsen  method,  104,  156 


— 


BRADBURY,    AGNEW,    &    CO.    LD.,    PRINTERS,   LONDON   AND   TONBRIDGE. 


COLUMBIA  UNIVERSITY  LIBRARIES 

This  book  is  due  on  the  date  indicated  below,  or  at  the 
expiration  of  a  definite  period  after  the  date  of  borrowing,  as 
provided  by  the  library  rules  or  by  special  arrangement  with 
the  Librarian  in  charge. 


DATE  BORROWED 


DATE    DUE 


DATE   BORROWED 


DATE    DUE 


m1  71950 


C28(842)MSC 


Tva 


fed  (o\ 


P\<\ 


